Immunomodulatory oligonucleotides

Oligonucleotides containing unthylated CpG dinucleotides and therapeutic utilities based on their ability to stimulate an immune response in a subject are disclosed. Also disclosed are therapies for treating diseases associated with immune system activation that are initiated by unthylated CpG dinucleotides in a subject comprising administering to the subject oligonucleotides that do not contain unmethylated CpG sequences (i.e. methylated CpG sequences or no CpG sequence) to outcompete unmethylated CpG nucleic acids for binding. Further disclosed are methylated CpG containing dinucleotides for use antisense therapies or as in vivo hybridization probes, and immunoinhibitory oligonucleotides for use as antiviral therapeutics.

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Description
RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent application Ser. No. 10/690,495 filed on Oct. 21, 2003 which a continuation of U.S. patent application Ser. No. 09/415,142, filed Oct. 9, 1999, now abandoned, which is a divisional of U.S. patent application Ser. No. 08/386,063, filed Feb. 7, 1995, now issued as U.S. Pat. No. 6,194,388 B1, which is a continuation-in-part of U.S. patent application Ser. No. 08/276,358, filed Jul. 15, 1994, now abandoned.

GOVERNMENT SUPPORT

The work resulting in this invention was supported in part by National Institute of Health Grant No. R29-AR42556-01. The U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

DNA Binds to Cell Membrane and is Internalized

In the 1970's, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R. A., W. Meinke, and D. A. Goldstein. 1971. “Membrane-associated DNA in the cytoplasm of diploid human lyphocytes”. Proc. Natl. Acad. Sci. USA 68: 1212; Agrawal, S. K., R. W. Wagner, P. K. McAllister, and B. Rosenberg. 1975. “Cell-surface associated nucleic acid in tumorigenic cells made visible with platinum-pyrimidine complexes by electron microscopy”. Proc. Natl. Acad. Sci. USA 72:928). In 1985 Bennett et al. presented the first evidence that DNA binding to lymphocytes is similar to a ligand receptor interaction: binding is saturable, competitive, and leads to DNA endocytosis and degradation (Bennett, R. M., G. T. Gabor, and M. M. Merritt, 1985. “DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA”. J. Clin. Invest. 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are able to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J. W., and J. S. Cohen 1991. “Cellular uptake of antisense oligodeoxynucleotides”. Advanced Drug Delivery Reviews 6:235; Akhtar, S., Y. Shoji, and R. L. Juliano, 1992. “Pharmaceutical aspects of the biological stability and membrane transport characteristics of antisense oligonucleotides”. In: Gene Regulation: Biology of Antisense RNA and DNA. R. P. Erickson, and J. G. Izant, eds. Raven Press, Ltd. New York, pp. 133; and Zhao, Q., T. Waldschmidt, E. Fisher, C. J. Herrera, and A. M. Krieg, 1994. “Stage specific oligonucleotide uptake in murine bone marrow B cell precursors”. Blood, 84:3660). No receptor for DNA or ODN uptake has yet been cloned, and it is not yet clear whether ODN binding and cell uptake occurs through the same or a different mechanism from that of high molecular weight DNA.

Lymphocyte ODN uptake has been shown to be regulated by cell activation. Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen Con A showed enhanced ODN uptake by T but not B cells (Krieg, A. M., F. Gmelig-Meyling, M. F. Gourley, W. J. Kisch, L. A. Chrisey, and A. D. Steinberg. 1991. “Uptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible”. Antisense Research and Development 1:161).

Immune Effects of Nucleic Acids

Several polynucleotides have been extensively evaluated as biological response modifiers. Perhaps the best example is poly (I, C) which is a potent inducer of IFN production as well as a macrophage activator and inducer of NK activity (Talmadge, J. E., J. Adams, H. Phillips, M. Collins, B. Lenz, M. Schneider, E. Schlick, R Ruffmann, R. H. Wiltrout, and M. A. Chirigos. 1985. “Immunomodulatory effects in mice of polyinosinic-polycytidylic acid complexed with poly-L:-lysine and carboxymethylcellulose”. Cancer Res. 45:1058; Wiltrout, R. H., R. R. Salup, T. A. Twilley, and J. E. Talmadge. 1985. “Immunomodulation of natural killer activity by polyribonucleotides”. J. Biol. Resp. Mod 4:512; Krown, S. E. 1986. “Interferons and interferon inducers in cancer treatment”. Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp, J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W. G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P. Creekmore. 1992. “Polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin 2 in patients with cancer: clinical and immunological effects”. Canc. Res. 52:3005). It appears that this murine NK activation may be due solely to induction of IFN-β secretion (Ishikawa, R., and C. A. Biron. 1993. “IFN induction and associated changes in splenic leukocyte distribution”. J. Immunol. 150:3713). This activation was specific for the ribose sugar since deoxyribose was ineffective. Its potent in vitro antitumor activity led to several clinical trials using poly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (to reduce degradation by RNAse) (Talmadge, J. E., et al., 1985. cited supra; Wiltrout, R. H., et al., 1985. cited supra); Krown, S. E., 1986. cited supra); and Ewel, C. H., et al., 1992. cited supra). Unfortunately, toxic side effects have thus far prevented poly (I,C) from becoming a useful therapeutic agent.

Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may replace “B cell differentiation factors” (Feldbush, T. L., and Z. K. Ballas. 1985. “Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation”. J. Immunol. 134:3204; and Goodman, M. G. 1986. “Mechanism of synergy between T cell signals and C8-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine”. J. Immunol. 136:3335). 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation of MHC restricted CTU (Feldbush, T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E. Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. “Activation of murine natural killer cells and macrophages by 8-bromoguanosine”. J. Immunol. 140:3249), and synergize with IL-2 in inducing murine LAK generation (Thompson, R. A., and Z. K. Ballas. 1990. “Lymphokine-activated killer (LAK) cells. V.8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation”. J. Immunol. 145:3524). The NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, R. A., et al. 1990. cited supra). Recently, a 5′ triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human γδ T cells (Constant, P., F. Davodeau, M.-A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and J.-J. Fournie. 1994. “Stimulation of human γδ T cells by nonpeptidic mycobacterial ligands” Science 264:267). This report indicated the possibility that the immune system may have evolved ways to preferentially respond to microbial nucleic acids.

Several observations suggest that certain DNA structures may also have the potential to activate lymphocytes. For example, Bell et al. reported that nucleosomal protein-DNA complexes (but not naked DNA) in spleen cell supernatants caused B cell proliferation and immunoglobulin secretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990. “Immunogenic DNA-related factors”. J. Clin. Invest. 85:1487). In other cases, naked DNA has been reported to have immune effects. For example, Messina et al. have recently reported that 260 to 800 bp fragments of poly (dG).(dC) and poly (dG.dC) were mitogenic for B cells (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1993. “The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens”. Cell. Immunol. 147:148). Tokunaga, et al. have reported that dG.dC induces γ-IFN and NK activity (Tokunaga, S. Yamamoto, and K Namba. 1988. “A synthetic single-stranded DNA, poly(dG,dC), induces interferon-α/β and -γ, augments natural killer activity, and suppresses tumor growth” Jpn. J. Cancer Res. 79:682). Aside from such artificial homopolymer sequences, Pisetsky et al. reported that pure mammalian DNA has no detectable immune effects, but that DNA from certain bacteria induces B cell activation and immunoglobulin secretion (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1991. “Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA”. J. Immunol. 147:1759). Assuming that these data did not result from some unusual contaminant, these studies suggested that a particular structure or other characteristic of bacterial DNA renders it capable of triggering B cell activation. Investigations of mycobacterial DNA sequences have demonstrated that ODN which contain certain palindrome sequences can activate NK cells (Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O. Yano, and T. Tokunaga. 1992. “Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity”. J. Immunol. 148:4072; Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino, S., Yamamoto, T. Yamamoto, T. Kataoka, and T. Tokunaga. 1992. “Oligonucleotide sequences required for natural killer-cell activation”. Jpn. J. Cancer Res. 83.1128).

Several phosphorothioate modified ODN have been reported to induce in vitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu, and W. E. Paul. 1992. “An antisense oligonucleotide complementary to a sequence in Iγ2b increases γ2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion”. J. Exp. Med. 175:597; Branda, R. F., A. L. Moore, L. Mathews, J. J. McCormack, and G. Zon. 1993. “Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1”. Biochem. Pharmacol. 45:2037; McIntyre, K. W., K. Lombard-Gillooly, J. R. Perez, C. Kunsch, U. M. Sarmiento, J. D. Larigan, K. T. Landreth, and R. Narayanan 1993. “A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-κ β T65 causes sequence-specific immune stimulation”. Antisense Res. Develop. 3:309; and Pisetsky, D. S., and C. F. Reich. 1993. “Stimulation of murine lymphocyte proliferation by a phosphorothioate oligonucleotide with antisense activity for herpes simplex virus”. Life Sciences 54:101). These reports do not suggest a common structural motif or sequence element in these ODN that might explain their effects.

The CREB/ATF Family of Transcription Factors and their Role in Replication

The cAMP response element binding protein (CREB) and activating transcription factor (ATF) or CREB/ATF family of transcription factors is a ubiquitously expressed class of transcription-factors of which 11 members have so far been cloned (reviewed in de Groot, R. P., and P. Sassone-Corsi: “Hormonal control of gene expression: Multiplicity and versatility of cyclic adenosine 3′,5′-monophosphate-responsive nuclear regulators”. Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson: “Transcriptional-regulation by CREB and its relatives”. Biochim. Biophys. Acta 1174:221, 1993.). They all belong to the basic region leucine zipper (bZip) class of proteins. All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral-transcription, but some splicing variants which lack the activation domain are inhibitory. CREB/ATF proteins can bind DNA as homo- or hetero-dimers through the cAMP response element, the CRE, the consensus form of which is the unmethylated sequence TGACGTC (binding is abolished if the CpG is methylated) (Iguchi-Ariga, S. M. M., and W. Schaffler: “CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation”. Genes & Develop. 3:612, 1989.).

The transcriptional activity of the CRE is increased during B cell activation (Xie, H. T. C. Chiles, and T. L. Rothstein: “Induction of CREB activity via the surface Ig receptor of B cells”. J. Immunol 151:880; 1993.). CREB/ATF proteins appear to regulate the expression of multiple genes through the CRE including immunologically important genes such as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W. R. Waterman, A. C. Webb, and P. E. Auron: “Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1β gene”. Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Herandez, D. Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: “Antisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice”. Cancer Res. 53:577, 1993), IFN-β (Du, W., and T. Maniatis: “An ATF/CREB binding site protein is required for virus induction of the human interferon B gene”. Proc. Natl. Acad. Sci. USA 89:2150, 1992), TGF-β1 (Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: “Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-B1 gene”. Biochim. Biophys. Acta 1219:55, 1994), TGF-β2, class II MHC (Cox, P. M., and C. R. Goding: “An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DRa promoter and activation by SV40 T-antigen”. Nucl. Acids Res. 20:4881, 1992.), E-selectin, GM-CSF, CD-8α, the germline Igα constant region gene, the TCR Vβ gene, and the proliferating cell nuclear antigen (Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M. B. Prystowskly: “Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes”. Mol. Cell. Biol. 14:4233, 1994.). In addition to activation through the cAMP-pathway, CREB can also mediate transciptional responses to changes in intracellular Ca++ concentration (Sheng, M., G. McFadden, and M. E. Greenberg: “Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB”. Neuron 4:571, 1990).

The role of protein-protein interactions in transcriptional activation by CREB/ATF proteins appears to be extremely important. Activation of CREB through the cyclic AMP pathway requires protein kinase A (PKA), which phosphorylates CREB341 on ser133 and allows it to bind to a recently cloned protein, CBP (Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G. Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman: “Nuclear protein CBP is a coactivator for the transcription factor CREB”. Nature 370:223, 1994; Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smea, M. Karin, J. Feramisco, and M. Montminy: “Activation of cAMP and mitogen responsive genes relies on a common nuclear factor”. Nature 370:226, 1994.). CBP in turn interacts with the basal transcription factor TFIIB causing increased transcription. CREB also has been reported to interact with dTAFII 110, a TATA binding protein-associated factor whose binding may regulate transcription (Ferreri, K., G. Gill, and M. Montminy: “The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex”. Proc. Natl. Acad. Sci. USA 91:1210, 1994.). In addition to these interactions, CREB/ATF proteins can specifically bind multiple other nuclear factors Hoeffler, J. P., J. W. Lustbader, and C.-Y. Chen: “Identification of multiple nuclear factors that interact with cyclic adenosine 3′,5′-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions”. Mol. Endocrinol. 5:256, 1991) but the biologic significance of most of these interactions is unknown. CREB is normally thought to bind DNA either as a homodimer or as a heterodimer with several other proteins. Surprisingly, CREB monomers constitutively activate transcription (Krajewski, W., and K. A. W. Lee: “A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator”. Mol. Cell. Biol. 14:7204, 1994.).

Aside from their critical role in regulating cellular transcription, it has recently been shown that CREB/ATF proteins are subverted by some infectious viruses and retroviruses, which require them for viral replication. For example, the cytomegalovirus immediate early promoter, one of the strongest known mammalian promoters, contains eleven copies of the CRE which are essential for promoter function (Chang, Y.-N., S. Crawford, J. Stall, D. R Rawlins, K.-T. Jeang, and G. S. Hayward: “The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements”, J. Virol. 64:264, 1990). At least some of the transcriptional activating effects of the adenovirus E1A protein, which induces many promoters, are due to its binding to the DNA binding domain of the CREB/ATF protein, ATF-2, which mediates E1A inducible transcription activation (Liu, F., and M. R. Green: “Promoter targeting by adenovirus E1a through interaction with different cellular DNA-binding domains”. Nature 368:520, 1994). It has also been suggested that E1A binds to the CREB-binding protein, CBP (Arany, Z., W. R. Sellers, D. M. Livingston, and R. Eckner: “E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators”. Cell 77:799, 1994). Human T lymphotropic virus-I (HTLV-1), the retrovirus which causes human T cell leukemia and tropical spastic paresis, also requires CREB/ATF proteins for replication. In this case, the retrovirus produces a protein, Tax, which binds to CREB/ATF proteins and redirects them from their normal cellular binding sites to different DNA sequences (flanked by G- and C-rich sequences) present within the HTLV transcriptional enhancer (Paca-Uccaralertkun, S., L.-J. Zhao, N. Adya, J. V. Cross, B. R. Cullen, I. N Boros, and C.-Z. Giam: “In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax”. Mol. Cell. Biol. 14:456, 1994; Adya, N., L.-J. Zhao, W. Huang, I. Boros, and C.-Z. Giam: “Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB”. Proc. Natl. Acad. Sci. USA 91:5642, 1994).

SUMMARY OF THE INVENTION

The instant invention is based on the finding that certain oligonucleotides containing unmethylated cytosine-guaine (CpG) dinucleotides activate lymphocytes as evidenced by in vitro and in vivo data. Based on this finding, the invention features, in one aspect, novel immunostimulatory oligonucleotide compositions.

In a preferred embodiment, an immunostimulatory oligonucleotide is synthetic, between 2 to 100 base pairs in size and contains a consensus mitogenic CpG motif represented by the formula:
5′X1X2CGX3X43′

    • wherein C and G are unmethylated, X1, X2, X3 and X4 are nucleotides and, a GCG trinucleotide sequence is not present at or near the 5′ and 3′ termini.

For facilitating uptake into cells, CpG containing immunostimulatory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides. Enhanced immunostimulatory activity has been observed where X1X2 is the dinucleotide GpA and/or X3X4 is the dinucleotide is most preferably TpC or also TpT. Further enhanced immunostimulatory activity has been observed where the consensus motif X1X2CGX3X4 is preceded on the 5′ end by a T.

In a second aspect, the invention features useful methods, which are based on the immunostimulatory activity of the oligonucleotides. For example, lymphocytes can either be obtained from a subject and stimulated ex vivo upon contact with an appropriate oligonucleotide; or a non-methylated CpG containing oligonucleotide can be administered to a subject to facilitate in vivo activation of a subject's lymphocytes. Activated lymphocytes, stimulated by the methods described herein (e.g. either ex vivo or in vivo), can boost a subject's immune response. The immunostimulatory oligonucleotides can therefore be used to treat, prevent or ameliorate an immune system deficiency (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection in a subject. In addition, immunostimulatory oligonucleotides can also be administered as a vaccine adjuvant, to stimulate a subject's response to a vaccine. Further, the ability of immunostimulatory cells to induce leukemic cells to enter the cell cycle, suggests a utility for treating leukemia by increasing the sensitivity of chronic leukemia cells and then administering conventional ablative chemotherapy.

In a third aspect, the invention features neutral oligonucleotides (i.e. oligonucleotide that do not contain an unmethylated CpG or which contain a methylated CpG dinucleotide). In a preferred embodiment, a neutralizing oligonucleotide is complementary to an immunostimulatory sequence, but contains a methylated instead of an unmethylated CpG dinucleotide sequence and therefore can compete for binding with unmethylated CpG containing oligonucleotides. In a preferred embodiment, the methylation occurs at one or more of the four carbons and two nitrogens comprising the cytosine six member ring or at one or more of the five carbons and four nitrogens comprising the guanine nine member double ring. 5′ methyl cytosine is a preferred methylated CpG.

In a fourth aspect, the invention features useful methods using the neutral oligonucleotides. For example, in vivo administration of neutral oligonucleotides should prove useful for treating diseases such as systemic lupus erythematosus, sepsis and autoimmune diseases, which are caused or exacerbated by the presence of unmethylated CpG dimers in a subject. In addition, methylation CpG containing antisense oligonucleotides or oligonucleotide probes would not initiate an immune reaction when administered to a subject in vivo and therefore would be safer than corresponding unmethylated oligonucleotides.

In a fifth aspect, the invention features immunoinhibitory oligonucleotides, which are capable of interfering with the activity of viral or cellular transcription factors. In a preferred embodiment, immunoinhibitory oligonucleotides are between 2 to 100 base pairs in size and contain a consensus immunoinhibitory CpG motif represented by the formula:
5′GCGXnGCG3′

wherein X=a nucleotide and n in the range of 0-50. In a preferred embodiment, X is a pyrimidine.

For facilitating uptake into cells, immunoinhibitory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides.

In a sixth and final aspect, the invention features various uses for immunoinhibitory oligonucleotides. Immunoinhibitory oligonucleotides have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted.

Other features and advantages of the invention will become more apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms and phrases shall have the meanings set forth below:

An “oligonucleotide” or “oligo” shall mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term “oligonucleotide” as used herein refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs). The term “oligonucleotide” shall also include oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).

A “stabilized oligonucleotide” shall mean an oligonucleotide that is relatively resistant to, in vivo degradation (e.g. via an exo- or endo-nuclease). Preferred stabilized oligonucleotides of the instant invention have a modified phosphate backbone. Especially preferred oligonucleotides have a posphorothioate modified phosphate backbone (i.e. at least one of the phosphate oxygens is replaced by sulfur). Other stabilized oligonucleotides include: nonionic-DNA analogs, such as alkyl- and aryl-phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.

An “immunostimulatory oligonucleotide”, “immunostimulatory CpG containing oligonucleotide”, or “CpG ODN” refer to an oligonucleotide, which contains a cytosine, guanine dinucleotide sequence and stimulates (e.g. has a mitogenic effect) on vertebrate lymphocyte. Preferred immunostimulatory oligonucleotides are between 2 to 100 base pairs in size and contain a consensus mitogenic CpG motif represented by the formula:
5′X1X2CGX3X43′

    • wherein C and G are unmethylated, X1, X2, X3 and X4 are nucleotides and a GCG trinucleotide sequence is not present at or near the 5′ and 3′ termini.

Preferably the immunostimulatory oligonucleotides range between 8 to 40 base pairs in size. In addition, the immunostimulatory oligonucleotides are preferably stabilized oligonucleotides, particularly preferred are phosphorothioate stabilized oligonucleotides. In one preferred embodiment, X1X2 is the dinucleotide GpA. In another preferred embodiment, X3X4 is preferably the dinucleotide TpC or also TpT. In a particularly preferred embodiment, the consensus motif X1X2CGX3X4 is preceded on the 5′ end by a T. Particularly preferred consensus sequences are TGACGTT or TGACGTC.

A “neutral oligonucleotide” refers to an oligonucleotide that does not contain an unmethylated CpG or an oligonucleotide which contains a methylated CpG dinucleotide. In a preferred embodiment, a neutralizing oligonucleotide is complementary to an immunostimulatory sequence, but contains a methylated instead of an unmethylated CpG dinucleotide sequence and therefore can compete for binding with unmethylated CpG containing oligonucleotides. In a preferred embodiment, the methylation occurs at one or more of the four carbons and two nitrogens comprising the cytosine six member ring or at one or more of the five carbons and four nitrogens comprising the guanine nine member double ring. 5′ methyl cytosine is a preferred methylated CpG.

An “immunoinhibitory oligonucleotide” or “immunoinhibitory CpG containing oligonucleotide” is an oligonucleotide that. Preferable immunoinhibitory oligonucleotides are between 2 to 100 base pairs in size and can be represented by the formula:
5′GCGXnGCG3′

wherein X=a nucleotide and n=in the range of 0-50. In a preferred embodiment, X is a pyrimidine.

For facilitating uptake into cells, immunoinhibitory oligonucleotides are preferably in the range of 8 to 40 base pairs in size. Prolonged immunostimulation can be obtained using stabilized-oligionucleotides, particularly phosphorothioate stabilized.

“Palindromic sequence” shall mean an inverted repeat (i.e. a sequence such as ABCDEE′D′C′B′A′ in which A and A′ are bases capable of forming the usual Watson-Crick base pairs). In vivo, such sequences may form double stranded structures.

An “oligonucleotide delivery complex” shall mean an oligonucleotide associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells). Examples of oligonucleotide delivery complexes include oligonucleotides associated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by target cell specific receptor). Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex should be cleavable under appropriate conditions within the cell so that the oligonucleotide is released in a functional form.

An “immune system deficiency” shall mean a disease or disorder in which the subject's immune system is not functioning in normal capacity or in which it would be useful to boost a subject's immune response for example to eliminate a tumor or cancer (e.g. tumors of the brain; lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes), fungal (e.g. Candida sp.), bacterial or parasitic (e.g. Leishmania, Toxoplasma) infection in a subject.

A “disease associated with immune system activation” shall mean a disease or condition caused or exacerbated by activation of the subject's immune system. Examples include systemic lupus erythematosus, sepsis and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

A “subject” shall mean a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, mouse, etc.

Certain Unmethylated CpG Containing Oligos have B Cell Stimulatory Activity as Shown In Vitro and In Vivo

In the course of investigating the lymphocyte stimulatory effects of two antisense oligonucleotides specific for endogenous retroviral sequences, using protocols described in the attached Examples 1 and 2, it was surprisingly found that two out of twenty-four “controls” (including various scrambled, sense, and mismatch controls for a panel of “antisense” ODN) also mediated B cell activation and IgM secretion, while the other “controls” had no effect.

Two observations suggested that the mechanism of this B cell activation by the “control” ODN may not involve antisense effects 1) comparison of vertebrate DNA sequences listed in GenBank showed no greater homology than that seen with non-stimulatory ODN and 2) the two controls showed no hybridization to Northern blots with 10 μg of spleen poly A+ RNA. Resynthesis of these ODN on a different synthesizer or extensive purification by polyacrylamide gel-electrophoresis or high pressure liquid chromatography gave identical stimulation, eliminating the possibility of an impurity. Similar stimulation was seen using B cells from C3H/HeJ mice, eliminating the possibility that lipopolysaccharide (LPS) contamination could account for the results.

The fact that two “control” ODN caused B cell activation similar to that of the two “antisense” ODN raised the possibility that all four ODN were stimulating B cells through some non-antisense mechanism involving a sequence motif that was absent in all of the other nonstimulatory control ODN. In comparing these sequences, it was discovered that all of the four stimulatory ODN contained ODN dinucleotides that were in a different sequence context from the nonstimulatory control.

To determine whether the CpG motif present in the stimulatory ODN was responsible for the observed stimulation, over 300 ODN ranging in length from 5 to 42 bases that contained methylated, unmethylated, or no CpG dinucleotides in various sequence contexts were synthesized. These ODNs, including the two original “controls” (ODN 1 and 2) and two originally synthesized as “antisense” (ODN 3D and 3M; Krieg A. M. J. Immunol. 143:2448 (1989)), were then examined for in vitro effects on spleen cells (representative sequences are listed in Table 1). Several ODN that contained CpG dinucleotides induced B cell activation and IgM secretion; the magnitude of this stimulation typically could be increased by adding more CpG dinucleotides (Table 1; compare ODN 2 to 2a or 3D to 3Da and 3Db). Stimulation did not appear to result from an antisense mechanism or impurity. ODN caused no detectable activation of γδ or other T cell populations.

Mitogenic ODN sequences uniformly became nonstimulatory if the CpG dinucleotide was mutated (Table. 1; compare ODN 1 to 1a; 3D to 3Dc; 3M to 3Ma; and 4 to 4a) or if the cytosine of the CpG dinucleotide was replaced by 5-methylcytosine (Table 1; ODN 1b, 2b, 2c, 3Dd, and 3Mb). In contrast, methylation of other cytosines did not reduce ODN activity (ODN 1c, 2d, 3De and 3Mc). These data confirmed that a CpG motif is the essential element present in ODN that activate B cells.

In the course of these studies, it became clear that the bases flanking the CpG dinucleotide played an important role in determining the B cell activation induced by an ODN. The optimal stimulatory motif was determined to consist of a CpG flanked by two 5′ purines (preferably a GpA dinucleotide) and two 3′ pyrimidines (preferably a TpT or TpC dinucleotide). Mutations of ODN to bring the CpG motif closer to this ideal improved stimulation (e.g. compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the motif reduced stimulation (e.g. compare ODN 3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutations outside the CpG motif did not reduce stimulation (e.g. compare ODN 1 to 1d; 3D to 3Dg; 3M to 3Me).

Of those tested, ODNs shorter than 8 bases were non-stimulatory (e.g. ODN 4e). Among the forty-eight 8 base ODN tested, the most stimulatory sequence identified was TCAACGTT (ODN 4) which contains the self complementary “palindrome” AACGTT. In further optimizing this motif, it was found that ODN containing Gs at both ends showed increased stimulation, particularly if the ODN were rendered nuclease resistant by phosphorothioate modification of the terminal internucleotide linkages. ODN 1585 (5′ GGGGTCAACGTTCAGGGGGG 3′ (SEQ ID NO:1)), in which the first two and last five internucleotide linkages are phosphorothioate modified caused an average 25.4 fold increase in mouse spleen cell proliferation compared to an average 3.2 fold increase in proliferation induced by ODN 1638, which has the same sequence as ODN 1585 except that the 10 Gs at the two ends are replaced by 10 As. The effect of the G-rich ends is cis; addition of an ODN with poly G ends but no CpG motif to cells along with 1638 gave no increased proliferation.

Other octamer ODN containing a 6 base palindrome with a TpC dinucleotide at the 5′ end were also active if they were close to the optimal motif (e.g. ODN 4b,4c). Other dinucleotides at the 5′ end gave reduced stimulation (eg ODN 4f; all sixteen possible dinucleotides were tested). The presence of a 3′ dinucleotide was insufficient to compensate for the lack of a 5′ dinucleotide (eg. ODN 4g). Disruption of the palindrome eliminated stimulation in octamer ODN (eg., ODN 4h), but palindromes were not required in longer ODN.

TABLE 1  Oligonucleotide Stimulation of B Cells Stimulation Index' ODN Sequence (5′ to 3')† 3H Uridine IgM Production 1(SEQ ID NO: 2) GCTAGACGTTAGCGT  6.1 ± 0.8 17.9 ± 3.6 la(SEQ ID NO: 3) ......T........  1.2 ± 0.2  1.7 ± 0.5 lb(SEQ ID NO: 4) ......Z........  1.2 ± 0.1  1.8 ± 0.0 lc(SEQ ID NO: 5) ............Z.. 10.3 ± 4.4  9.5 ± 1.8 ld(SEQ ID NO: 6) ..AT......GAGC. 13.0 ± 2.3 18.3 ± 7.5 2(SEQ ID NO: 7) ATGGAAGGTCCAGCGTTCTC  2.9 ± 0.2 13.6 ± 2.0 2a(SEQ ID NO: 8) ..C..CTC..G.........  7.7 ± 0.8 24.2 ± 3.2 2b(SEQ ID NO: 9) ..Z..CTC.ZG..Z......  1.6 ± 0.5  2.8 ± 2.2 2c(SEQ ID NO: 10) ..Z..CTC..G.........  3.1 ± 0.6  7.3 ± 1.4 2d(SEQ ID NO: 11) ..C..CTC..G......Z..  7.4 ± 1.4 27.7 ± 5.4 2e(SEQ ID NO: 12) ............A.......  5.6 ± 2.0 ND 3D(SEQ ID NO: 13) GAGAACGCTGGACCTTCCAT  4.9 ± 0.5 19.9 ± 3.6 3Da(SEQ ID NO: 14) .........C..........  6.6 ± 1.5 33.9 ± 6.8 3Db(SEQ ID NO: 15) .........C.......G.. 10.1 ± 2.8 25.4 ± 0.8 3Dc(SEQ ID NO: 16) ...C.A..............  1.0 ± 0.1  1.2 ± 0.5 3Dd(SEQ ID NO: 17) .....Z..............  1.2 ± 0.2  1.0 ± 0.4 3De(SEQ ID NO: 18) .............Z......  4.4 ± 1.2 18.8 ± 4.4 3Df(SEQ ID NO: 19) .......A............  1.6 ± 0.1  7.7 ± 0.4 3Dg(SEQ ID NO: 20) .........CC.G.ACTG..  6.1 ± 1.5 18.6 ± 1.5 3M (SEQ ID NO: 21) TCCATGTCGGTCCTGATGCT  4.1 ± 0.2   23.2 ± 4.9 3Ma(SEQ ID NO: 22) ......CT............  0.9 ± 0.1  1.8 ± 0.5 3Mb(SEQ ID NO: 23) .......Z............  1.3 ± 0.3  1.5 ± 0.6 3Mc(SEQ ID NO: 24) ...........Z........  5.4 ± 1.5  8.5 ± 2.6 3Md(SEQ ID NO: 25) ......A..T.......... 17.2 ± 9.4 ND 3Me(SEQ ID NO: 26) .........CC.G.ACTG..  3.6 ± 0.2 14.2 ± 5.2 4 TCAACGTT  6.1 ± 1.4 19.2 ± 5.2 4a ....GC..  1.1 ± 0.2  1.5 ± 1.1 4b ...GCGC.  4.5 ± 0.2  9.6 ± 3.4 4c ...TCGA.  2.7 ± 1.0 ND 4d ..TT..AA  1.3 ± 0.2 ND 4e -.......  1.3 ± 0.2  1.1 ± 0.5 4f C.......  3.9 ± 1.4 ND 4g --......CT  1.4 ± 0.3 ND 4h .......C  1.2 ± 0.2 ND LPS  7.8 ± 2.5  4.8 ± 1.0 'Stimulation indexes are the means and std. dev. derived from at least 3 sep- arate experiments, and are compared to wells cultured with no added ODN.-ND = not done. CpG dinucleotides are underlined. Dots indicate identity; dashes indicate deletions. Z indicates 5 methyl cytosine.)

The kinetics of lymphocyte activation were investigated using mouse spleen cells. When the cells were pulsed at the same time as ODN addition and harvested just four hours later, there was already a two-fold increase in 3H uridine incorporation. Stimulation peaked at 12-48 hours and then decreased. After 24 hours, no intact ODN were detected, perhaps accounting for the subsequent fall in stimulation when purified B cells with or without anti-IgM (at a submitogenic dose) were cultured with CpG ODN, proliferation was found to synergistically increase about 10-fold by the two mitogens in combination after 48 hours. The magnitude of stimulation was concentration dependent and consistently exceeded that of LPS under optimal conditions for both. Oligonucleotides containing a nuclease resistant phosphorothioate backbone were approximately two hundred times more potent than unmodified oligonucleotides.

Cell cycle analysis was used to determine the proportion of B cells activated by CpG-ODN. CpG-ODN induced cycling in more than 95% of B cells (Table 2). Splenic B lymphocytes sorted by flow cytometry into CD23-(marginal zone) and CD23+ (follicular) subpopulations were equally responsive to ODN-induced stimulation, as were both resting and activated populations of B cells isolated by fractionation over Percoll gradients. These studies demonstrated that CpG-ODN induce essentially all B cells to enter the cell cycle.

TABLE 2 Cell Cycle Analysis with CpG ODN Percent of cells in Treatment G0 G1 SA + G2 + M Media 97.6 2.4 0.02 ODN 1a 95.2 4.8 0.04 ODN 1d 2.7 74.4 22.9 ODN 3Db 3.5 76.4 20.1 LPS (30 μg/ml) 17.3 70.5 12.2

The mitogenic effects of CpG ODN on human cells, were tested on peripheral blood mononuclear cells (PBMCs) obtained from two patients with chronic lymphocytic leukemia (CLL), as described in Example 1. Control ODN containing no CpG dinucleotide sequence showed no effect on the basal proliferation of 442 cpm and 874 cpm (proliferation measured by 3H thymidine incorporation) of the human cells. However, a phosphorothioate modified CpG ODN 3Md (SEQ ID NO: 25) induced increased proliferation of 7210 and 86795 cpm respectively in the two patients at a concentration of just 1 μM. Since these cells had been frozen, they may have been less responsive to the oligos than fresh cells in vivo. In addition, cells from CLL patients typically are non-proliferating, which is why traditional chemotherapy is not effective.

Certain B cell lines such as WEHI-231 are induced to undergo growth arrest and/or apoptosis in response to crosslinking of their antigen receptor by anti-IgM (Jakway, J. P. et al., “Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide and other bacterial products” J. Immunol. 137:2225 (1986); Tsubata. T., J. Wu and T. Honjo: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40.” Nature 364: 645 (1993)). WEHI-231 cells are rescued from this growth arrest by certain stimuli such as LPS and by the CD40 ligand. ODN containing the CpG motif were also found to protect WEHI-231 from anti-IgM induced growth arrest, indicating that accessory cell populations are not required for the effect.

To better understand the immune effects of unmethylated CpG ODN, the levels of cytokines and prostaglandins in vitro and in vivo were measured. Unlike LPS, CpG ODN were not found to induce purified macrophages to produce prostaglandin PGE2. In fact, no apparent direct effect of CpG ODN was detected on either macrophages or T cells. In vivo or in whole spleen cells, no significant increase in the following interleukins: IL-2, IL-3, IL-4, or IL-10 was detected within the first six hours. However, the level of IL-6 increased strikingly within 2 hours in the serum of mice injected with CpG ODN. Increased expression of IL-12 and interferon gamma (IFN-γ) by spleen cells was also detected within the first two hours.

To determine whether CpG ODN can cause in vivo immune stimulation, DBA/2 mice were injected once intraperitoneally with PBS or phosphorothioate CpG or non-CpG ODN at a dose of 33 mg/kg (approximately 500 μg/mouse). Pharmacokinetic studies in mice indicate that this dose of phosphorothioate gives levels of approximately 10 μg/g in spleen tissue (within the effective concentration range determined from the in vitro studies described herein) for longer than twenty-four hours (Agrawal, S. et al. (1991) Proc. Natl. Acad. Sci. USA 91:7595). Spleen cells from mice were examined twenty-four hours after ODN injection for expression of B cells surface activation markers Ly-6A/E, Bla-1, and class II MHC using three color flow cytometry and for their spontaneous proliferation using 3H thymidine: Expression of all three activation markers was significantly increased in B cells from mice injected with CpG ODN, but not from mice injected with PBS or non-CpG ODN. Spontaneous 3H thymidine incorporation was increased by 2-6 fold in spleen cells from mice injected with the stimulatory ODN compared to PBS or non-CpG ODN-injected mice. After 4 days, serum IgM levels in mice injected with CpG ODN in vivo were increased by approximately 3-fold compared to controls. Consistent with the inability of these agents to activate T cells, there was minimal change in T cell expression of the IL-2R or CD-44.

Degradation of phophodiester ODN in serum is predominantly mediated by 3′ exonucleases, while intracellular ODN degradation is more complex, involving 5′ and 3′ exonucleases and endonucleases. Using a panel of ODN bearing the 3D sequence with varying numbers of phosphorothioate modified linkages at the 5′ and 3′ ends, it was empirically determined that two 5′ and five 3′ modified linkages are required to provide optimal stimulation with this CpG ODN.

Unmethylated CpG Containing Oligos have NK Cell Stimulatory Activity

As described in flier detail in Example 4, experiments were conducted to determine whether CpG containing oligonucleotides stimulated the activity of natural killer (NK) cells in addition to B cells. As shown in Table 3, a marked induction of NY, activity among spleen cells cultured with CpG ODN 1 and 3Dd was observed. In contrast, there was relatively no induction in effectors that had been treated with non-CpG control ODN.

TABLE 3 Induction Of NK Activity By CpG Oligodeoxynucleotides (ODN) % YAC-1 Specific Lysis* % 2C11 Specific Lysis Effector: Target Effector: Target ODN 50:1 100:1 50:1 100:1 None -1.1 -1.4 15.3 16.6 1 16.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0 non-CpG ODN -1.6 -1.7 14.8 15.4

Neutralizing Activity of Methylated CpG Containing Oligos

B cell mitogenicity of ODN in which cytosines in CpG motifs or elsewhere were replaced by 5-methylcytosine were tested as described in Example 1. As shown in Table 1 above, ODN containing methylated CpG motifs were non-mitogenic (Table 1; ODN 1c, 2f, 3De, and 3Mc). However, methylation of cytosines other than in a CpG dinucleotide retained their stimulatory properties (Table 1, ODN 1d, 2d, 3Df, and 3Md).

Immunoinhibitory Activity of Oligos Containing a GCG Trinucleotide Sequence at or Near Both Termini

In some cases, ODN containing CpG dinucleotides that are not in the stimulatory motif described above were found to block the stimulatory effect of other mitogenic CpG ODN. Specifically the addition of an atypical CpG motif consisting of a GCG near or at the 5′ and/or 3′ end of CpG ODN actually inhibited stimulation of proliferation by other CpG motifs. Methylation or substitution of the cytosine in a GCG motif-reverses this effect. By itself, a GCG motif in an ODN has a modest mitogenic effect, though far lower that that seen with the preferred CpG motif.

Proposed Mechanisms of Action of Immunostimulatory, Neutralizing and Immunoinhibitory Oligonucleotides

Unlike antigens that trigger B cells through their surface Ig receptor, CpG-ODN did not induce any detectable Ca2+ flux, changes in protein tyrosine phosphorylation, or IP 3 generation. Flow cytometry with FITC-conjugated ODN with or without a CpG motif was performed as described in Zhao, Q et al., (Antisense Research and Development 3:53-66 (1993)), and showed equivalent membrane binding, cellular uptake, efflux, and intracellular localization. This suggests that there may not be cell membrane proteins specific for CpG ODN. Rather than acting through the cell membrane, that data suggests that unmethylated CpG containing oligonucleotides require cell uptake for activity: ODN covalently linked to a solid Teflon support were nonstimulatory, as were biotinylated ODN immobilized on either avidin beads or avidin coated petri dishes. CpG ODN conjugated to either FITC or biotin retained full mitogenic properties, indicating no steric hindrance.

The optimal CpG motif (TGACGTT/C is identical to the CRE (cyclic AMP response element). Like the mitogenic effects of CpG ODN, binding of CREB to the CRE is abolished if the central CpG is methylated. Electrophoretic mobility shift assays were used to determine whether CpG ODN, which are single stranded, could compete with the binding of B cell CREB/ATF proteins to their normal binding site, the doublestranded CRE. Competition assays demonstrated that single stranded ODN containing CpG motifs could completely compete the binding of CREB to its binding site, while ODN without CpG motifs could not. These data support the conclusion that CpG ODN exert their mitogenic effects through interacting with one or more B cell CREB/ATF proteins in some way. Conversely, the presence of GCG sequences or other atypical CPG motifs near the 5′ and/or 3′ ends of ODN likely interact with CREB/ATF proteins in a way that does not cause activation, and may even prevent it.

The stimulatory CpG motif is common in microbial genomic DNA, but quite rare in vertebrate DNA. In addition, bacterial DNA has been reported to induce B cell proliferation and immunoglobulin (Ig) production, while mammalian DNA does not (Messina, J. P. et al., J. Immunol. 147:1759 (1991)). Experiments further described in Example 3, in which methylation of bacterial DNA with CpG methylase was found to abolish mitogenicity, demonstrates that the difference in CpG status is the cause of B cell stimulation by bacterial DNA. This data supports the following conclusion: that unmethylated CpG dinucleotides present within bacterial DNA are responsible for the stimulatory effects of bacterial DNA.

Teleologically, it appears likely that lymphocyte activation by the CpG motif represents an immune defense mechanism that can thereby distinguish bacterial from host DNA. Host DNA would induce little or no lymphocyte activation due to it CpG suppression and methylation. Bacterial DNA would cause selective lymphocyte activation in infected tissues. Since the CpG pathway synergizes with B cell activation through the antigen receptor, B cells bearing antigen receptor specific for bacterial antigens would receive one activation signal through cell membrane Ig and a second signal from bacterial DNA, and would therefore tend to be preferentially activated. The interrelationship of this pathway with other pathways of B cell activation provide a physiologic mechanism employing a polyclonal antigen to induce antigen-specific responses.

Method for Making Immunostimulatory Oligos

For use in the instant invention, oligonucleotides can be synthesized de novo using any of a number of procedures well known in the art. For example, the β-cyanoethyl phosphoramidite method (S. L. Beaucage and M. H. Caruthers, (1981) Tet. Let. 22:1859); nucleoside H-phosphonate method (Garegg et al., (1986) Tet. Let. 27: 4051-4054; Froehler et al., (1986) Nucl. Acid. Res. 14: 5399-5407; Garegg et al., (1986) Tet. Let. 27:4055-4058, Gaffney et al., (1988) Tet. Let. 29:2619-2622). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market. Alternatively, oligonucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.

For use in vivo, oligonucleotides are preferably relatively resistant to degradation (e.g. via endo- and exo-nucleases). Oligonucleotide stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized oligonucleotide has a phosphorothioate modified backbone. The pharmacokinetics of phosphorothioate ODN show that they have a systemic half-life of forty-eight hours in rodents and suggest that they may be useful for in vivo applications (Agrawal, S. et al. (1991) Proc. Natl. Acad. Sci. USA 88:7595). Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H phosphonate chemistries. Aryl- and alkyl-phosphonates can be made e.g. (as described in U.S. Pat. No. 4,469,863); and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. and Peyman, A. (1990) Chem. Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165).

For administration in vivo, oligonucleotides may be associated with a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form an “oligonucleotide delivery complex”. Oligonucleotides can be ionically, or covalently associated with appropriate molecules using techniques which are well known in the art. A variety of coupling or crosslinking agents can be used e.g. protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Oligonucleotides can alternatively be encapsulated in liposomes or virosomes using well-known techniques.

The present invention is further illustrated by the following Examples which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

Therapeutic Uses of Immunostimulatory Oligos

Based on their immunostimulatory properties, oligonucleotides containing at least-one unmethylated CpG dinucleotide can be administered to a subject in vivo to treat an “immune system deficiency”. Alternatively, oligonucleotides containing at least one unmethylated CpG dinucleotide can be contacted with lymphocytes (e.g. B cells or NK cells) obtained from a subject having an immune system deficiency ex vivo and activated lymphocytes can then be reimplanted in the subject.

Immunostimulatory oligonucleotides can also be administered to a subject in conjunction with a vaccine, as an adjuvant, to boost a subject's immune system to effect better response from the vaccine. Preferably the unmethylated CpG dinucleotide is administered slightly before or at the same time as the vaccine.

Preceding chemotherapy with an immunostimulatory oligonucleotide should prove useful for increasing the responsiveness of the malignant cells to subsequent chemotherapy. CpG ODN also increased natural killer cell activity in both human and murine cells. Induction of NK activity may likewise be beneficial in cancer immunotherapy.

Therapeutic Uses for Neutral Oligonucleotides

Oligonucleotides that are complementary to certain target sequences can be synthesized and administered to a subject in vivo. For example, antisense oligonucleotides hybridize to complementary mRNA, thereby preventing expression of a specific target gene. The sequence-specific effects of antisense oligonucleotides have made them useful research tools for the investigation of protein function. Phase I/II human trials of systemic antisense therapy are now underway for acute myelogenous leukemia and HIV.

In addition, oligonucleotide probes (i.e. oligonucleotides with a detectable label) can be administered to a subject to detect the presence of a complementary sequence based on detection of bound label. In vivo administration and detection of oligonucleotide probes may be useful for diagnosing certain diseases that are caused or exacerbated by certain DNA sequences (e.g. systemic lupus erythematosus, sepsis and autoimmune diseases).

Antisense oligonucleotides or oligonucleotide probes in which any or all CpG dinucleotide is methylated, would not produce an immune reaction when administered to a subject in vivo and therefore would be safer than the corresponding non-methylated CpG containing oligonucleotide.

For use in therapy, an effective amount of an appropriate oligonucleotide alone or formulated as an oligonucleotide delivery complex can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells (e.g. B-cells and NK cells). Preferred routes of administration include oral and transdermal (e.g. via a patch). Examples of other routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.). The injection can be in a bolus or a continuous infusion.

An oligonucleotide alone or as an oligonucleotide delivery complex can be administered in conjunction with a pharmaceutically acceptable carrier. As used herein, the phrase “pharmaceutically acceptable carrier” is intended to include substances that can be coadministered with an oligonucleotide or an oligonucleotide delivery complex and allows the oligonucleotide to perform its intended function. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the oligonucleotides falls within the scope of the instant invention.

The language “effective amount” of an oligonucleotide refers to that amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of an oligonucleotide containing at least one methylated CpG for treating an immune system deficiency could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal infection. An effective amount for use as a vaccine adjuvant could be that amount useful for boosting a subject's immune response to a vaccine. An “effective amount” of an oligonucleotide lacking a non-methylated CpG for use in treating a disease associated with immune system activation, could be that amount necessary to outcompete non-methylated CpG containing nucleotide sequences. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular oligonucleotide being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular oligonucleotide without necessitating undue experimentation.

The studies reported above indicate that unmethylated CpG containing oligonucleotides are directly mitogenic for lymphocytes (e.g. B cells and NK cells). Together, with the presence of these sequences in bacterial DNA, these results suggest that the underrepresentation of CpG dinucleotides in animal genomes, and the extensive methylation of cytosines present in such dinucleotides, may be explained by the existence of an immune defense mechanism that can distinguish bacterial from host DNA. Host DNA would commonly be present in many anatomic regions and areas of inflammation due to apoptosis (cell death), but generally induces little or no lymphocyte activation. However, the presence of bacterial DNA containing unmethylated CpG motifs can cause lymphocyte activation precisely in infected anatomic regions, where it is beneficial. This novel activation pathway provides a rapid alternative to T cell dependent antigen specific B-cell activation. However, it is likely that B cell activation would not be totally nonspecific. B cells bearing antigen receptors specific for bacterial products could receive one activation signal through cell membrane Ig, and a second from bacterial DNA, thereby more vigorously triggering antigen specific immune responses.

As with other immune defense mechanisms, the response to bacterial DNA could have undesirable consequences in some settings. For example, autoimmune responses to self antigens would also tend to be preferentially triggered by bacterial infections, since autoantigens could also provide a second activation signal to autoreactive B cells triggered by bacterial DNA. Indeed the induction of autoimmunity by bacterial infections is a common clinical observance. For example, the autoimmune disease systemic lupus erythematosus, which is: i) characterized by the production of anti-DNA-antibodies; ii) induced by drugs which inhibit DNA methyltransferase (Cornacchia, E. J. et al., J. Clin. Invest. 92:38 (1993)); and iii) associated with reduced DNA methylation (Richardson, B., L. et al., Arth. Rheum 35:647 (1992)), is likely triggered at least in part by activation of DNA-specific B cells through stimulatory signals provided by CpG motifs, as well as by binding of bacterial DNA to antigen receptors.

Further, sepsis, which is characterized by high morbidity and mortality due to massive and nonspecific activation of the immune system may be initiated by bacterial DNA and other products released from dying bacteria that reach concentrations sufficient to directly activate many lymphocytes.

Lupus, sepsis and other “diseases associated with immune system activation” may be treated, prevented or ameliorated by administering to a subject oligonucleotides lacking an unmethylated CpG dinucleotide (e.g. oligonucleotides that do not include a CpG motif or oligonucleotides in which the CpG motif is methylated) to block the binding of unmethylated CpG containing nucleic acid sequences. Oligonucleotides lacking an unmethylated CpG motif can be administered alone or in conjunction with compositions that block an immune cell's reponse to other mitogenic bacterial products (e.g. LPS).

The following serves to illustrate-mechanistically how oligonucleotides containing an unmethylated CpG dinucleotide can treat, prevent or ameliorate the disease lupus. Lupus is commonly thought to be triggered by bacterial or viral infections. Such infections have been reported to stimulate the production of nonpathogenic antibodies to single stranded DNA. These antibodies likely recognize primarily bacterial sequences including unmethylated CpGs. As disease develops in lupus, the anti-DNA antibodies shift to pathogenic antibodies that are specific for double-stranded DNA. These antibodies would have increased binding for methylated CpG sequences and their production would result from a breakdown of tolerance in lupus. Alternatively, lupus may result when a patient's DNA becomes hypomethylated, thus allowing anti-DNA antibodies specific for unmethylated CpGs to bind to self DNA and trigger more widespread autoimmunity through the process referred to is “epitope spreading”.

In either case, it may be possible to restore tolerance in lupus patients by coupling antigenic oligonucleotides to a protein carrier such as gamma globulin (IgG). Calf-thymus DNA complexed to gamma globulin has been reported to reduce anti-DNA antibody formation.

Therapeutic Uses of Oligos Containing GCG Trinucleotide Sequences at or Near Both Termini

Based on their interaction with CREB/ATF, oligonucleotides containing GCG trinucleotide sequences at or near both termini have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted. Based on this activity, an effective amount of inhibitory oligonucleotides can be administered to a subject to treat or prevent a viral infection.

EXAMPLES Example 1 Effects of ODNs on B Cell Total RNA Synthesis and Cell Cycle

B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA/2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) (“B cells”). B cells contained fewer than 1% CD4+ or CD8+ cells. 8×104 B cells were dispensed in triplicate into 96 well-microtiter-plates in 100 μl RPMI containing 10% FBS (heat inactivated to 65° C. for 30 min.), 50 μM 2-mercaptoethanol, 100 U/mil penicillin, 100 ug/ml streptomycin, and 2 mM L-glutamate. 20 μM ODN were added at the start of culture for 20 h at 37° C., cells pulsed with 1 μCi of 3H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ELISA spot assay after culture of whole spleen cells with ODN at 20 μM for 48 hr. Data, reported in Table 1, represent the stimulation index compared to cells cultured without ODN. Cells cultured without ODN gave 687 cpm, while cells cultured with 20 μg/ml LPS (determined by titration to be the optimal concentration) gave 99,699 cpm in this experiment 3H thymidine incorporation assays showed similar results, but with some nonspecific inhibition by thymidine released from degraded ODN (Matson. S and A. M. Krieg (1992) Nonspecific suppression of 3H-thymidine incorporation by control oligonucleotides. Antisense Research and Development 2:325).

For cell cycle analysis, 2×106 B cells-were cultured for 48 hr. in 2 ml tissue culture medium alone, or with 30 μg/ml LPS or with the indicated phosphorothioate modified ODN at 1 μM. Cell cycle analysis was performed as described in (Darzynkiewicz, Z. et al., Proc. Natl. Acad. Sci. USA 78:2881 (1981)).

To test the mitogenic effects of CpG ODN on human cells, perpheral blood monocyte cells (PBMCs) were obtained from two patients with chronic lymphocytic leukemia (CLL) a disease in which the circulating cells are malignant B cells. Cells were cultured for 48 hrs and pulsed for 4 hours with tritiated thymidine as described above.

Example 2 Effects of ODN on Production of IgM from B Cells

Single cell suspensions from the spleens of freshly killed mice were treated with anti-Thyl, anti-CD4, and anti-CD8 and complement by the method of Leibson et al., J. Exp. Med. 154:1681 (1981)). Resting B cells (<02% T cell contamination) were isolated from the 63-70% band of a discontinuous Percoll gradient by the procedure of DeFranco et al, J. Exp. Med. 155:1523 (1982). These were cultured as described above in 30 μM ODN or 20 μg/ml LPS for 48 hr. The number of B cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay (Klinman, D. M. et al. J. Immunol. 144:506 (1990)). In that assay, B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The Ig they produced (>99% IgM) was detected using phosphatase-labelled anti-Ig (Southern Biotechnology Associated, Birmingham, Ala.). The antibodies produced by individual B cells were visualized by addition of BCIP (Sigma Chemical Co., St.; Louis Mo.) which forms an insoluble blue precipitate in the presence of phosphatase. The dilution of cells producing 20-40 spots/well was used to determine the total-number of antibody-secreting B cells/sample. All assays were performed in triplicate. In some experiments, culture supernatants were assayed for IgM by ELISA, and showed similar increases in response to CpG-ODN.

Table 1

Example 3 B Cell Stimulation by Bacterial DNA

DBA/2B cells were cultured with no DNA or 50 μg/ml of a) Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NFS/N mouse spleen genomic DNAs for 48 hours, then pulsed with 3H thymidine for 4 hours prior to cell harvest. Duplicate DNA samples were digested with DNAse I for 30 minutes at 37 C prior to addition to cell cultures. E coli DNA also induced an 8.8 fold increase in the number of IgM secreting B cells by 48 hours using the ELISA-spot assay.

DBA/2B cells were cultured with either no additive, 50 μg/ml LPS or the ODN 1; 1a; 4; or 4a at 20 uM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20, 40 or 80 μM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were <5%.

Example 4 Effects of ODN on Natural Killer (NK) Activity

10×106 C57BL/6 spleen cells were cultured in two ml RPMI (supplemented as described for Example 1) with or without 40 μM CpG or non-CpG ODN for forty-eight hours. Cells were washed, and then used as effector cells in a short term 51Cr release assay with YAC-1 and 2C11, two NK sensitive target cell lines (Ballas, Z. K. et al. (1993) J. Immunol. 150:17). Effector cells were added at various concentrations to 104 51Cr-labeled target cells in V-bottom microtiter plates in 0.2 ml, and incubated in 5% CO2 for 4 hr. at 37° C. Plates were then centrifuged, and an aliquot of the supernatant counted for radioactivity. Percent specific lysis was determined by calculating the ratio of the 51Cr released in the presence of effector cells minus the 51Cr released when the target cells are cultured alone, over the total counts released after cell lysis in 2% acetic acid minus the 51Cr cpm released when the cells are cultured alone.

Example 5 In Vivo Studies with CpG Phosphorothioate ODN

Mice were weighed and injected IP with 0.25 ml of sterile PBS or the indicated phophorothioate ODN dissolved in PBS. Twenty four hours later, spleen cells were harvested, washed, and stained for flow cytometry using phycoerythrin conjugated 6B2 to ate on B cells in conjunction with biotin conjugated anti Ly-6A/E or anti-Iad (Pharmingen, San Diego, Calif.) or anti-Bla-1 (Hardy., R. R. et al., J. Exp. Med. 159:1169 (1984). Two mice were studied for each condition and analyzed individually.

Example 6 Titration of Phosphorothioate ODN for B Cell Stimulation

B cells were cultured with phosphorothioate ODN with the sequence of control ODN 1a or the CpG ODN 1d and 3Db and then either pulsed after 20 hr with 3H uridine or after 44 hr with 3H thymidine before harvesting and determining cpm.

Example 7 Rescue of B Cells from Apoptosis

WEHI-231 cells (5×104/well) were cultured for 1 hr. at 37 C. in the presence or absence of LPS or the control ODN 1a or the CpG ODN 1d and 3Db before addition of anti-IgM (1 μ/ml). Cells were cultured for a further 20 hr. before a 4 hr. pulse with 2 μCi/well 3H thymidine. In this experiment, cells with no ODN or anti-IgM gave 90.4×103 by addition of anti-IgM. The phosphodiester ODN shown in Table 1 gave similar protection, though with some nonspecific suppression due to ODN degradation. Each experiment was repeated at least 3 times with similar results.

Example 8 In Vivo Induction of IL-6

DBA/2 female mice (2 mos. old) were injected IP with 500 μg CpG or control phosphorothioate ODN. At various time points after injection, the mice were bled. Two mice were studied for each time point. IL-6 was measured by Elisa, and IL-6 concentration was calculated by comparison to a standard curve generated using recombinant IL-6. The sensitivity of the assay was 10 pg/ml. Levels were undetectable after 8 hr.

Example 9 Binding of B Cell CREB/ATF to a Radiolabelled Doublestranded CRE Probe (CREB)

Whole cell extracts from CH12.LX B cells showed 2 retarded bands when analyzed by EMSA with the CRE probe (free probe is off the bottom of the figure). The CREB/ATF protein(s) binding to the CRE were competed by the indicated amount of cold CRE, and by single-stranded CpG ODN, but not by non-CpG ODN.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for stimulating a subject's response to a vaccine comprising administering an immunostimulatory oligonucleotide adjuvant as a vaccine adjuvant with the vaccine to the subject to stimulate the subject's response to the vaccine, wherein the immunostimulatory oligonucleotide comprises a phosphate backbone modification and greater than two unmethylated cytosine-guanine dinucleotides, and wherein the oligonucleotide is at least eight nucleotides in length, wherein the oligonucleotide is linked to a nucleic acid delivery complex.

2. The method of claim 1, wherein the nucleic acid delivery complex is a cationic lipid.

3. The method of claim 1, wherein the oligonucleotide is covalently linked to the nucleic acid delivery complex.

4. The method of claim 1, wherein the oligonucleotide is ionically linked to or encapsulated in the nucleic acid delivery complex.

5. The method of claim 1, wherein the nucleic acid delivery complex is a sterol.

6. A method for stimulating a subject's response to a vaccine comprising administering an immunostimulatory oligonucleotide adjuvant as a vaccine adjuvant with the vaccine to the subject to stimulate the subject's response to the vaccine, wherein the immunostimulatory oligonucleotide comprises a phosphate backbone modification and an unmethylated cytosine-guanine dinucleotide, wherein the oligonucleotide is at least eight nucleotides in length and wherein the unmethylated cytosine-guanine dinucleotide is flanked by two 5′ purines and two 3′ pyrimidines.

7. The method of claim 6, wherein the oligonucleotide includes at least two unmethylated cytosine-guanine motifs.

8. The method of claim 7, wherein at least one of the at least two unmethylated cytosine-guanine motifs is not palindromic.

Referenced Cited
U.S. Patent Documents
2215233 September 1940 Ruskin
3627874 December 1971 Vella et al.
3906092 September 1975 Hilleman et al.
3911117 October 1975 Ender
3914450 October 1975 Robbins et al.
4188375 February 12, 1980 Straub
4544559 October 1, 1985 Gil et al.
4741914 May 3, 1988 Kimizuka et al.
4758553 July 19, 1988 Ogoshi
4806376 February 21, 1989 Saeki et al.
4956296 September 11, 1990 Fahnestock
4963387 October 16, 1990 Nakagawa et al.
4994442 February 19, 1991 Gil et al.
5066500 November 19, 1991 Gil et al.
5231085 July 27, 1993 Alexander et al.
5234811 August 10, 1993 Beutler et al.
5268365 December 7, 1993 Rudolph et al.
5288509 February 22, 1994 Potman et al.
5488039 January 30, 1996 Masor et al.
5492899 February 20, 1996 Masor et al.
5585479 December 17, 1996 Hoke et al.
5591721 January 7, 1997 Agrawal et al.
5602109 February 11, 1997 Masor et al.
5612060 March 18, 1997 Alexander
5650156 July 22, 1997 Grinstaff et al.
5663153 September 2, 1997 Hutcherson et al.
5679647 October 21, 1997 Carson et al.
5684147 November 4, 1997 Agrawal et al.
5700590 December 23, 1997 Masor et al.
5712256 January 27, 1998 Kulkarni et al.
5723335 March 3, 1998 Hutcherson et al.
5756353 May 26, 1998 Debs
5780448 July 14, 1998 Davis
5786189 July 28, 1998 Locht et al.
5840705 November 24, 1998 Tsukada
5849719 December 15, 1998 Carson et al.
5895652 April 20, 1999 Giampapa
5922766 July 13, 1999 Acosta et al.
5929226 July 27, 1999 Padmapriya
5976580 November 2, 1999 Ivey et al.
5980958 November 9, 1999 Naylor et al.
6004534 December 21, 1999 Langer et al.
6022853 February 8, 2000 Kuberasampath et al.
6031086 February 29, 2000 Switzer
6090791 July 18, 2000 Sato et al.
6174872 January 16, 2001 Carson et al.
6191257 February 20, 2001 Ledley et al.
6194388 February 27, 2001 Krieg et al.
6207646 March 27, 2001 Krieg et al.
6214806 April 10, 2001 Krieg et al.
6218371 April 17, 2001 Krieg et al.
6221882 April 24, 2001 Macfarlane
6225292 May 1, 2001 Raz et al.
6239116 May 29, 2001 Krieg et al.
6248720 June 19, 2001 Mathiowitz et al.
6339068 January 15, 2002 Krieg et al.
6339630 January 15, 2002 Hartley
6399630 June 4, 2002 Macfarlane
6406705 June 18, 2002 Davis et al.
6426336 July 30, 2002 Carson et al.
6429199 August 6, 2002 Krieg et al.
6479504 November 12, 2002 Macfarlane et al.
6498147 December 24, 2002 Nerenberg et al.
6498148 December 24, 2002 Raz
6503533 January 7, 2003 Korba
6514948 February 4, 2003 Raz et al.
6521637 February 18, 2003 Macfarlane
6534062 March 18, 2003 Raz et al.
6544518 April 8, 2003 Gerard et al.
6552006 April 22, 2003 Raz et al.
6558670 May 6, 2003 Friede et al.
6562798 May 13, 2003 Schwartz
6589940 July 8, 2003 Raz et al.
6610661 August 26, 2003 Carson et al.
6653292 November 25, 2003 Krieg et al.
6727230 April 27, 2004 Hutcherson et al.
6737066 May 18, 2004 Moss
6821957 November 23, 2004 Krieg et al.
6943240 September 13, 2005 Bauer et al.
6949520 September 27, 2005 Hartmann et al.
6977245 December 20, 2005 Klinman et al.
7001890 February 21, 2006 Wagner et al.
7223741 May 29, 2007 Krieg
7271156 September 18, 2007 Krieg et al.
7354711 April 8, 2008 Macfarlane
7402572 July 22, 2008 Krieg et al.
7410975 August 12, 2008 Lipford et al.
7488490 February 10, 2009 Davis et al.
7517861 April 14, 2009 Krieg et al.
7524828 April 28, 2009 Krieg et al.
7534772 May 19, 2009 Weiner et al.
7566703 July 28, 2009 Krieg et al.
7569553 August 4, 2009 Krieg
7576066 August 18, 2009 Krieg
7585847 September 8, 2009 Bratzler et al.
7605138 October 20, 2009 Krieg
7615539 November 10, 2009 Krieg et al.
7666674 February 23, 2010 Klinman et al.
7674777 March 9, 2010 Krieg et al.
7713529 May 11, 2010 Krieg et al.
7723022 May 25, 2010 Krieg et al.
7723500 May 25, 2010 Krieg et al.
7749979 July 6, 2010 Chaung et al.
7776344 August 17, 2010 Hartmann et al.
7795235 September 14, 2010 Krieg et al.
7807803 October 5, 2010 Krieg
7820379 October 26, 2010 Bauer et al.
7879810 February 1, 2011 Krieg et al.
7919477 April 5, 2011 Klinman et al.
7935351 May 3, 2011 Klinman et al.
7935675 May 3, 2011 Krieg et al.
7951786 May 31, 2011 Klinman et al.
7956043 June 7, 2011 Krieg et al.
8003115 August 23, 2011 Fearon et al.
8008266 August 30, 2011 Krieg et al.
8017749 September 13, 2011 Das Gupta et al.
8021834 September 20, 2011 O'Hagan et al.
8030285 October 4, 2011 Klinman et al.
8034802 October 11, 2011 Averett
8043622 October 25, 2011 Klinman et al.
8053422 November 8, 2011 Klinman et al.
8058249 November 15, 2011 Krieg et al.
8114418 February 14, 2012 Fearon et al.
8114419 February 14, 2012 Krieg
8114848 February 14, 2012 Krieg et al.
8124590 February 28, 2012 Van Nest et al.
8129351 March 6, 2012 Krieg et al.
8148340 April 3, 2012 Krieg et al.
8158592 April 17, 2012 Krieg et al.
8188254 May 29, 2012 Uhlmann et al.
8202688 June 19, 2012 Davis et al.
8222225 July 17, 2012 Klinman et al.
20010044416 November 22, 2001 McCluskie et al.
20010046967 November 29, 2001 Van Nest
20020028784 March 7, 2002 Van Nest
20020055477 May 9, 2002 Nest
20020086295 July 4, 2002 Raz et al.
20020086839 July 4, 2002 Raz et al.
20020091097 July 11, 2002 Bratzler et al.
20020098199 July 25, 2002 Nest et al.
20020107212 August 8, 2002 Van Nest et al.
20020142978 October 3, 2002 Raz et al.
20020156033 October 24, 2002 Bratzler et al.
20020164341 November 7, 2002 Davis et al.
20020165178 November 7, 2002 Schetter et al.
20020198165 December 26, 2002 Bratzler et al.
20030022852 January 30, 2003 Van Nest et al.
20030026782 February 6, 2003 Krieg
20030026801 February 6, 2003 Weiner et al.
20030027782 February 6, 2003 Carson et al.
20030049266 March 13, 2003 Fearon et al.
20030050261 March 13, 2003 Krieg et al.
20030050263 March 13, 2003 Krieg et al.
20030050268 March 13, 2003 Krieg et al.
20030055014 March 20, 2003 Bratzler
20030059773 March 27, 2003 Van Nest et al.
20030078223 April 24, 2003 Raz et al.
20030091599 May 15, 2003 Davis et al.
20030092663 May 15, 2003 Raz et al.
20030100527 May 29, 2003 Krieg et al.
20030104044 June 5, 2003 Semple et al.
20030109469 June 12, 2003 Carson et al.
20030119773 June 26, 2003 Raz et al.
20030125292 July 3, 2003 Semple et al.
20030129251 July 10, 2003 Van Nest et al.
20030133988 July 17, 2003 Fearon et al.
20030139364 July 24, 2003 Krieg et al.
20030143213 July 31, 2003 Raz et al.
20030147870 August 7, 2003 Raz et al.
20030148316 August 7, 2003 Lipford et al.
20030148976 August 7, 2003 Krieg et al.
20030166001 September 4, 2003 Lipford
20030175731 September 18, 2003 Fearon et al.
20030181406 September 25, 2003 Schetter et al.
20030186921 October 2, 2003 Carson et al.
20030191079 October 9, 2003 Krieg et al.
20030199466 October 23, 2003 Fearon et al.
20030203861 October 30, 2003 Carson et al.
20030212026 November 13, 2003 Krieg et al.
20030212028 November 13, 2003 Raz et al.
20030216340 November 20, 2003 Van Nest et al.
20030224010 December 4, 2003 Davis et al.
20030232074 December 18, 2003 Lipford et al.
20030232780 December 18, 2003 Carson et al.
20030232856 December 18, 2003 MacFarlane
20040006010 January 8, 2004 Carson et al.
20040006034 January 8, 2004 Raz et al.
20040009949 January 15, 2004 Krieg
20040030118 February 12, 2004 Wagner et al.
20040053880 March 18, 2004 Krieg
20040067902 April 8, 2004 Bratzler et al.
20040067905 April 8, 2004 Krieg
20040087534 May 6, 2004 Krieg et al.
20040087538 May 6, 2004 Krieg et al.
20040092468 May 13, 2004 Schwartz
20040092472 May 13, 2004 Krieg
20040106568 June 3, 2004 Krieg et al.
20040131628 July 8, 2004 Bratzler et al.
20040132685 July 8, 2004 Krieg et al.
20040142469 July 22, 2004 Krieg et al.
20040143112 July 22, 2004 Krieg et al.
20040147468 July 29, 2004 Krieg et al.
20040152649 August 5, 2004 Krieg
20040152656 August 5, 2004 Krieg et al.
20040152657 August 5, 2004 Krieg et al.
20040162258 August 19, 2004 Krieg et al.
20040162262 August 19, 2004 Krieg et al.
20040167089 August 26, 2004 Krieg et al.
20040171150 September 2, 2004 Krieg et al.
20040171571 September 2, 2004 Krieg et al.
20040181045 September 16, 2004 Krieg et al.
20040198680 October 7, 2004 Krieg
20040198688 October 7, 2004 Krieg et al.
20040229835 November 18, 2004 Krieg et al.
20040234512 November 25, 2004 Wagner et al.
20040235770 November 25, 2004 Davis et al.
20040235774 November 25, 2004 Bratzler et al.
20040235777 November 25, 2004 Wagner et al.
20040235778 November 25, 2004 Wagner et al.
20040266719 December 30, 2004 McCluskie et al.
20050004061 January 6, 2005 Krieg et al.
20050004062 January 6, 2005 Krieg et al.
20050009774 January 13, 2005 Krieg et al.
20050031638 February 10, 2005 Dalemans et al.
20050032734 February 10, 2005 Krieg et al.
20050032736 February 10, 2005 Krieg et al.
20050037403 February 17, 2005 Krieg et al.
20050037985 February 17, 2005 Krieg et al.
20050043529 February 24, 2005 Davis et al.
20050049215 March 3, 2005 Krieg et al.
20050049216 March 3, 2005 Krieg et al.
20050054601 March 10, 2005 Wagner et al.
20050054602 March 10, 2005 Krieg et al.
20050059619 March 17, 2005 Krieg et al.
20050059625 March 17, 2005 Krieg et al.
20050070491 March 31, 2005 Krieg et al.
20050075302 April 7, 2005 Hutcherson et al.
20050100983 May 12, 2005 Bauer et al.
20050101554 May 12, 2005 Krieg et al.
20050101557 May 12, 2005 Krieg et al.
20050119273 June 2, 2005 Lipford et al.
20050123523 June 9, 2005 Krieg et al.
20050130911 June 16, 2005 Uhlmann et al.
20050148537 July 7, 2005 Krieg et al.
20050158336 July 21, 2005 Diamond et al.
20050169888 August 4, 2005 Hartmann et al.
20050171047 August 4, 2005 Krieg et al.
20050181422 August 18, 2005 Bauer et al.
20050182017 August 18, 2005 Krieg
20050196411 September 8, 2005 Moss
20050197314 September 8, 2005 Krieg et al.
20050215500 September 29, 2005 Krieg et al.
20050215501 September 29, 2005 Lipford et al.
20050233995 October 20, 2005 Krieg et al.
20050233999 October 20, 2005 Krieg et al.
20050239732 October 27, 2005 Krieg et al.
20050239733 October 27, 2005 Jurk et al.
20050239734 October 27, 2005 Uhlmann et al.
20050239736 October 27, 2005 Krieg et al.
20050244379 November 3, 2005 Krieg et al.
20050244380 November 3, 2005 Krieg et al.
20050245477 November 3, 2005 Krieg et al.
20050250726 November 10, 2005 Krieg et al.
20050256073 November 17, 2005 Lipford et al.
20050267057 December 1, 2005 Krieg
20050267064 December 1, 2005 Krieg et al.
20050277604 December 15, 2005 Krieg et al.
20050277609 December 15, 2005 Krieg et al.
20060003955 January 5, 2006 Krieg et al.
20060003962 January 5, 2006 Ahluwalia et al.
20060019916 January 26, 2006 Krieg et al.
20060019923 January 26, 2006 Davis et al.
20060058251 March 16, 2006 Krieg et al.
20060089326 April 27, 2006 Krieg et al.
20060094683 May 4, 2006 Krieg et al.
20060140875 June 29, 2006 Krieg et al.
20060154890 July 13, 2006 Bratzler et al.
20060172966 August 3, 2006 Lipford et al.
20060188913 August 24, 2006 Krieg et al.
20060211639 September 21, 2006 Bratzler et al.
20060211644 September 21, 2006 Krieg et al.
20060229271 October 12, 2006 Krieg et al.
20060241076 October 26, 2006 Uhlmann et al.
20060246035 November 2, 2006 Ahluwalia et al.
20060286070 December 21, 2006 Hartmann et al.
20060287263 December 21, 2006 Davis et al.
20070009482 January 11, 2007 Krieg et al.
20070010470 January 11, 2007 Krieg et al.
20070037767 February 15, 2007 Bratzler et al.
20070065467 March 22, 2007 Krieg et al.
20070066553 March 22, 2007 Krieg et al.
20070066554 March 22, 2007 Krieg et al.
20070078104 April 5, 2007 Krieg et al.
20070129320 June 7, 2007 Davis et al.
20070142315 June 21, 2007 Forsbach et al.
20070184465 August 9, 2007 Wagner et al.
20070202128 August 30, 2007 Krieg et al.
20070224210 September 27, 2007 Krieg et al.
20070232622 October 4, 2007 Lipford et al.
20080009455 January 10, 2008 Krieg et al.
20080026011 January 31, 2008 Krieg et al.
20080031936 February 7, 2008 Krieg et al.
20080045473 February 21, 2008 Uhlmann et al.
20080113929 May 15, 2008 Lipford et al.
20080226649 September 18, 2008 Schetter et al.
20090017021 January 15, 2009 Davis et al.
20090060927 March 5, 2009 Wagner et al.
20090117132 May 7, 2009 Readett et al.
20090142362 June 4, 2009 Krieg et al.
20090155212 June 18, 2009 Bratzler et al.
20090155307 June 18, 2009 Davis et al.
20090191188 July 30, 2009 Krieg et al.
20090202575 August 13, 2009 Krieg et al.
20090214578 August 27, 2009 Bauer
20090306177 December 10, 2009 Uhlmann et al.
20090311277 December 17, 2009 Krieg
20100125101 May 20, 2010 Krieg et al.
20100183639 July 22, 2010 Uhlmann et al.
20100285041 November 11, 2010 Uhlmann et al.
Foreign Patent Documents
1141740 February 1997 CN
1169434 January 1998 CN
1211443 March 1999 CN
0 178 267 April 1986 EP
0 468 520 January 1992 EP
0 216 133 July 1993 EP
0 302 758 March 1994 EP
2692897 December 1993 FR
2 216 416 November 1989 GB
56-008307 January 1981 JP
60-120962 June 1985 JP
62-025960 February 1987 JP
62-148428 July 1987 JP
62224259 October 1987 JP
8051953 February 1996 JP
8187059 July 1996 JP
9019276 January 1997 JP
10108655 April 1998 JP
91/05815 August 1991 WO
91/01327 September 1991 WO
WO 93/15207 August 1993 WO
94/02471 March 1994 WO
WO95/26204 October 1995 WO
WO96/02555 February 1996 WO
WO 97/12633 April 1997 WO
WO 97/28259 August 1997 WO
WO97/42975 November 1997 WO
WO 98/16247 April 1998 WO
WO98/49348 November 1998 WO
WO 99/11275 March 1999 WO
WO99/37151 July 1999 WO
WO 99/56755 November 1999 WO
WO 99/62923 December 1999 WO
WO 00/06588 February 2000 WO
WO 00/20039 April 2000 WO
WO 00/21556 April 2000 WO
WO 00/62787 October 2000 WO
WO 01/02007 January 2001 WO
WO 01/12223 February 2001 WO
WO 01/12804 February 2001 WO
WO 01/55341 August 2001 WO
WO 01/68077 September 2001 WO
WO 01/68078 September 2001 WO
WO 01/68103 September 2001 WO
WO 01/68116 September 2001 WO
WO 01/68117 September 2001 WO
WO 2004/007743 January 2004 WO
WO 2004/026888 April 2004 WO
WO 2004/094671 November 2004 WO
Other references
  • Mutwiri et al, J. Controlled Release, 2004, 97:1-17.
  • Weiner, J. Leukoc. Biol, 2000, 68:455-463.
  • Homer et al, Clinical Immunology, 2000, 95/1:S19-S29.
  • Klinman et al, Immunological Reviews, 2004, 199:201-216.
  • Klinman, Nature Reviews (Immunology), Apr. 2004, 4:1-10.
  • Verthelyi, In: Methods in Molecular Medicine, vol. 127: DAN Vaccines: Methods and Protocols: 2nd edition, pp. 139-158.
  • Freytag et al, Vaccines, 2005, 23:1804-1813.
  • Eastcott et al, Vaccine, 2001, 19:1636-1642.
  • Wernette et al, Vet. Immunol. and Immunopathol., 2002, 84:223-236.
  • McCluskie et al, Vaccine, 2000, 18:231-237.
  • Eriksson et al, Current Opinion in Immunology, 2002, 14:666-672.
  • Pink et al, Vaccine, 2004, 22:2097-2102.
  • Harandi et al, Current Opinion in Investigational Drugs, 2004, 5/2:141-146.
  • McCluskie et al, Vaccine, 2001, 19:3759-3768.
  • Gallichen et al, J. Immunology, 2001, 168:3451-3457.
  • McCluskie et al, J. Immunology, 1998, 161:4463-4466.
  • Weiner et al, PNAS USA, 1997, 94:10833-10837.
  • Hancock et al, Vaccine, 2001, 19:4874-4882.
  • O'Hagan et al, Biomolecular Engineering, 2001, 18:69-85.
  • Holmgren et al, Expert Rev. Vaccines, 2003, 2/2:205-217.
  • Manning et al, Experimental Gerontology, 2001, 37:107-126.
  • Dalpke et al, International J. Medical Microbiology, 2004, 294:345-354.
  • Krieg, Vaccine, 2001, 19:618-622.
  • McCluskie et al, Critical Rev. Immunology, 2001, 21:103-120.
  • Singh et al, Pharmaceutical Research, 2001, 18/10:1476-1479.
  • Toka et al, Immunological Reviews, 2004, 199:100-112.
  • McCluskie et al, Immunology Letters, 1999, 69/1:30-31 Abstract only.
  • Jiang et al, Infection and Immunity, 2003, 71/1:40-46.
  • Ellis, Vaccine, 2001, 19:2681-2687.
  • McCluskie et al, Vaccine, 2001, 19:2657-2660.
  • McCluskie et al, Vaccine, 2001, 19:413-422.
  • Roman et al, Nature Medicine, 1997, 3/8:849-854.
  • Threadgill et al, Vaccine, 1998, 16/1:76-82.
  • Leibson et al., Role of gamma-interferon in antibody-producing responses. Nature. Jun. 28-Jul. 4, 1984;309(5971):799-801.
  • Whalen et al., DNA-mediated immunization to the hepatitis B surface antigen. Activation and entrainment of the immune response. Ann N Y Acad Sci. Nov. 27, 1995;772:64-76.
  • Yamamoto, Cytokine production inducing action of oligo DNA. Rinsho Meneki. 1997; 29(9):1178-84. Japanese.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 3 (for judgment based on failure to comply with 35 U.S.C. 135(b)). (Electronically filed, unsigned). Jun. 7, 2004.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 4 (for judgment of no interference in fact). (Electronically filed, unsigned). Jun. 7, 2004.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 5 (for judgment based on lack of enablement). (Electronically filed, unsigned). Jun. 7, 2004.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 6 (for judgment based on lack of adequate written description). (Electronically filed, unsigned). Jun. 7, 2004.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 7 (motion to redefine interference to designate claims as not corresponding to the Count). (Electronically filed, unsigned). Jun. 7, 2004.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 8 (contingent motion to redefine the Count). (Electronically filed, unsigned). Jun. 7, 2004.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 9 (motion for benefit of earlier application). (Electronically filed, unsigned). Jun. 7, 2004.
  • Patent Interference No. 105,171. Iowa Preliminary Motion 10 (contingent motion to redefine the interference by adding a continuation application). (Electronically filed, unsigned). Jul. 2, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 3 (to Iowa Preliminary Motion 3 for judgment under 35 USC 135(b)). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 4 (to Iowa Preliminary Motion 4 for judgment of no interference in fact). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 5 (to Iowa Preliminary Motion 5 for judgment that UC's claim is not enabled). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 6 (to Iowa Preliminary Motion 6 for judgment based on lack of adequate written description). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 7 (to Iowa Preliminary Motion 7 to redefine the interference). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 8 (to Iowa Preliminary Motion 8 to redefine the Count). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Response 9 (to Iowa Contingent Motion 9 for benefit). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 10 (to Iowa Contingent Motion 10 to redefine the interference). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Opposition 11 (to Iowa Contingent Motion 11 to suppress). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 3 (in support of Iowa Preliminary Motion 3 for judgment under 35 U.S.C. §135(b)) (Electronically filed, unsigned). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 4 (in support of Iowa Preliminary Motion for judgment of no interference in fact) (Electronically filed, unsigned). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 5 (in support of Iowa Preliminary Motion 5 for judgment that UC's claim 205 is not enabled) (Electronically filed, unsigned). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 6 (in support of Iowa Preliminary Motion 6 for judgment based on lack of adequate written description) (Electronically filed, unsigned). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 7 (in support of Iowa Preliminary Motion 7 to redefine the interference) (Electronically filed, unsigned). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 8 (in support of Iowa Preliminary Motion 8 to redefine the count) (Electronically filed, unsigned). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 10 (in support of Iowa Preliminary Motion 10 to redefine the interference) (Electronically filed, unsigned). Oct. 15, 2004.
  • Patent Interference No. 105,171. Iowa Reply 11 (in support of Iowa Miscellaneous Motion to suppress). (Electronically filed, unsigned). Oct. 18, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Preliminary Statement. Jun. 7, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 1 (to designate additional claims of Iowa patent as corresponding to the Count). Jun. 7, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 2 (for judgment based on lack of written description support and introducing new matter). Jun. 7, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 3 (for judgment based on anticipation). Jun. 7, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 4 (for judgment based on obviousness). Jun. 7, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 5 (for judgment based on anticipation). Jun. 7, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 6 (for judgment based on inequitable conduct). Jun. 7, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Contingent Preliminary Motion 7 (for benefit of an earlier application under 37 CFR 1.633(j)). Jul. 2, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Contingent Preliminary Motion 8 (to add additional claims under 37 CFR 1.633(c)(2) and (i)). Jul. 2, 2004.
  • Amended Claims for U.S. Appl. No. 09/265,191, filed Mar. 10, 1999.
  • Patent Interference No. 105,171. Iowa Opposition 1 (opposition to motion to designate additional claims as corresponding to the Count) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent interference No. 105,171. Iowa Opposition 2 (opposition to motion for judgment based on lack of written description support and introducing new matter) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent Interference No. 105,171. Iowa Opposition 3 (opposition to motion for judgment based on anticipation) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent Interference No. 105,171. Iowa Opposition 4 (opposition to motion for judgment based on obviousness) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent Interference No. 105,171. Iowa Opposition 5 (opposition to motion for judgment based on anticipation) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent Interference No. 105,171. Iowa Opposition 6 (opposition to motion for judgment based on inequitable conduct) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent Interference No. 105,171. Iowa Opposition 7 (opposition to motion for benefit of an earlier application under 7 CFR 1.633(j)) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent Interference No. 105,171. Iowa Opposition 8 (opposition to motion to add additional claims under 37 CFR 1.633 (2) and (i)) (Electronically filed, unsigned). Sep. 9, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 1 (to Iowa's opposition to UC's motion to designate Iowa claims as corresponding to the Count). Oct. 15, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 2 (to Iowa's opposition to UC Preliminary Motion 2 for Judgment). Oct. 15, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 3 (to Iowa's Opposition to UC Preliminary Motion 3 for Judgment). Oct. 15, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 4 (to Iowa's Opposition to UC Preliminary Motion 4 for Judgment). Oct. 15, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 5 (to Iowa's Opposition to UC Preliminary Motion 5 for Judgment). Oct. 15, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 6 (to Iowa's opposition to UC Preliminary Motion 6 for judgment). Oct. 15, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 7 (to Iowa's Opposition to UC Preliminary Motion 7 for Benefit). Oct. 15, 2004.
  • Patent Interference No. 105,171. Regents of the University of California Reply 8 (to Iowa's Opposition to UC Preliminary Motion 8 to add additional claims). Oct. 15, 2004.
  • Patent Interference No. 105,171. Decision on Motion under 37 CFC §41.125. Mar. 10, 2005.
  • Patent Interference No. 105,171. Judgment and Order. Mar. 10, 2005.
  • Patent Interference No. 105,171. Regents of the University of California. Brief of Appellant. Jul. 5, 2005.
  • Patent Interference No. 105,171. University of Iowa and Coley Pharmaceutical Group, Inc. Brief of Appellees. Aug. 17, 2005.
  • Patent Interference No. 105,171. Regents of the University of California. Reply Brief of Appellant. Sep. 6, 2005.
  • Patent Interference No. 105,171. Regents of the University of California. Decision of CAFC. Jul. 17, 2006.
  • Ballas et al., Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol. Sep. 1, 1996;157(5):1840-5.
  • Branda et al., Amplification of antibody production by phosphorothioate oligodeoxynucleotides. J Lab Clin Med. Sep. 1996;128(3):329-38.
  • Carson et al., Oligonucleotide adjuvants for T helper 1 (Th1)-specific vaccination. J Exp Med. Nov. 17, 1997;186(10):1621-2.
  • Chace et al., Bacterial DNA-induced NK cell IFN-gamma production is dependent on macrophage secretion of IL-12. Clin Immunol Immunopathol. Aug. 1997;84(2):185-93.
  • Cowdery et al., Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases. The toxicity of lipopolysaccharides. J Immunol. Jun. 15, 1996;156(12):4570-5.
  • Davis et al., Plasmid DNA expression systems for the purpose of immunization. Cuff Opin. Biotechnol. Oct. 1997;8(5):635-46.
  • Gallichan et al., Specific secretory immune responses in the female genital tract following intranasal immunization with a recombinant adenovirus expressing glycoprotein B of herpes simplex virus. Vaccine Nov. 1995;13(16):1589-95.
  • Gaston et al., CpG methylation has differential effects on the binding of YYI and ETS proteins to the bi-directional promoter of the Surf-1 and Surf-2 genes. Nucleic Acids Res. Mar. 25, 1995;23(6):901-9.
  • Halpern et al., Bacterial DNA induces murine interferon-gamma production by stimulation of interleukin-12 and tumor necrosis factor-alpha. Cell Immunol. Jan. 10, 1996;167(1):72-8.
  • Higgins et al., Direct linkage of immunostimulatory DNA to a variety of proteins dramatically enhances Th1 and CTL responses. On Vaccine Research. National Foundation for Infectious Diseases (NFID) 5th Annual Conference. May 6-8, 2002. Abstract S4.
  • Kataoka et al., Immunotherapeutic potential in guinea-pig tumor model of deoxyribonucleic acid from Mycobacterium bovis BCG complexed with poly-L-lysine and carboxymethylcellulose. Jpn J Med Sci Biol. Oct. 1990;43(5):171-82.
  • Klinman et al., CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc Natl Acad Sci U S A. Apr. 2, 1996;93(7):2879-83.
  • Krieg et al., Lymphocyte activation mediated by oligodeoxynucleotides or DNA containing novel un-methylated CpG motifs. American College of Rheumatology 58th National Scientific Meeting. Minneapolis, Minnesota, Oct. 22, 1994. Abstracts. Arthritis Reum. Sep. 1994;37(9 Suppl).
  • Krieg et al., Phosphorothioate oligodeoxynucleotides: antisense or anti-protein? Antisense Res Dev. 1995 Winter;5(4):241.
  • Krieg, CpG DNA: a pathogenic factor in systemic lupus erythematosus? J Clin Immunol. Nov. 1995;15(6):284-92.
  • Krieg et al., Modification of antisense phosphodiester oligodeoxynucleotides by a 5′ cholesteryl moiety increases cellular association and improves efficacy. Proc Natl Acad Sci U S A. Feb. 1, 1993;90(3):1048-52.
  • Krieg, An innate immune defense mechanism based on the recognition of CpG motifs in microbial DNA. J Lab Clin Med. Aug. 1996;128(2):128-33.
  • Krieg et al., 1996 Meeting on Molecular Approaches to the Control of Infectious Diseases. Cold Spring Harbor Laboratory, Sep. 9-13, 1996: 116.
  • Krieg et al., Infection. In McGraw Hill Book. 1996:.242-3.
  • Krieg et al., Lymphocyte activation by CpG dinucleotide motifs in prokaryotic DNA. Trends Microbiol. Feb. 1996;4(2):73-6.
  • Kuramoto et al., Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injections of a Bacillus Calmette-Guerin nucleic acid fraction. Cancer Immunol Immunother. 1992;34(5):283-8.
  • Kuramoto et al., Changes of host cell infiltration into Meth A fibrosarcoma tumor during the course of regression induced by injections of a BCG nucleic acid fraction. Int J Immunopharmacol. Jul. 1992;14(5):773-82.
  • Kuramoto et al., In situ infiltration of natural killer-like cells induced by intradermal injection of the nucleic acid fraction from BCG. Microbiol Immunol. 1989;33(11):929-40.
  • Pisetsky et al, The immunologic properties of DNA. J Immunol. Jan. 15, 1996;156(2):421-3.
  • Pisetsky et al., Immunological properties of bacterial DNA. Ann N Y Acad Sci. Nov. 27, 1995;772:152-63.
  • Pisetsky, Immunologic consequences of nucleic acid therapy. Antisense Res Dev. 1995 Fall;5(3):219-25.
  • Pisetsky et al., Stimulation of in vitro proliferation of murine lymphocytes by synthetic oligodeoxynucleotides. Mol Biol Rep. Oct. 1993;18(3):217-21.
  • Pisetsky et al., Immune activation by bacterial DNA: a new genetic code. Immunity. Oct. 1996;5(4):303-10.
  • Rynkiewicz et al., Marked enhancement of antibody response to anthrax vaccine adsorbed with CPG 7909 in healthy volunteers. Intersci. Conf. Antimicrob. Agents Chemother. Poster (2005).
  • Sidman et al., Gamma-interferon is one of several direct B cell-maturing lymphokines. Nature. Jun. 28-Jul. 4, 1984;309(5971):801-4.
  • Sonehara et al., Hexamer palindromic oligonucleotides with 5′-CG-3′ motif(s) induce production of interferon. J Interferon Cytokine Res. Oct. 1996;16(10):799-803.
  • Stein et al., Problems in interpretation of data derived from in vitro and in vivo use of antisense oligodeoxynucleotides. Antisense Res Dev. 1994 Summer;4(2):67-9.
  • Wyatt et al. Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion. Proc Natl Acad Sci U S A. Feb. 15, 1994;91(4):1356-60.
  • Yi et al., Rapid immune activation by CpG motifs in bacterial DNA. Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway. J Immunol. Dec. 15, 1996;157(12):5394-402.
  • Yi et al., IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides. J Immunol. Jan. 15, 1996;156(2):558-64.
  • Yi et al., CpG DNA rescue of murine B lymphoma cells from anti-IgM-induced growth arrest and programmed cell death is associated with increased expression of c-myc and bcl-xL. J Immunol. Dec. 1, 1996;157(11):4918-25.
  • Anfossi et al. (P.N.A.S., 86, 9, 3379-83, 89, HCAPLUS, AN 1989:475562).
  • Azad, Raana F. et al., “Antiviral Activity of a Phosphorothioate Oligonucleotide Complementary to RNA of the Human Cytomegalovirus Major Immediate-Early Region,” Antimicrobial Agents and Chemotherapy, (1993) 37: 1945-1954.
  • Azuma, I., “Biochemical and Immunological Studies on Cellular Components of Tubercle bacilli,” Kekkaku (1992) 67(9):45-55.
  • Blaxter et al, “Genes expressed in Brugia malayi infective third stage larvae,” Molecular and Biochemical Parasitology, (1996) 77:77-93.
  • Etchart et al. “Class I-restricted CTL induction by mucosal immunization with naked DNA encoding measles virus haemagglutinin” pp. 15775761 vol. 72, 1998.
  • Ettinger, “Carrier Sequence Selection—One Key to Successful Vaccines,” Immunology Today, (1992) 13(2):52-55.
  • Fox, RI., “Mechanism of Action of Hydroxychloroquine as an antirheumatic Drug,” Chemical Abstracts (1994) 120:15, Abstract No. 182630.
  • Kataoka T, et al., “Antitumor Activity of Synthetic Oligonucleotides with Sequences from cDNA Encoding Proteins of Mycobacterium bovis BCG,” Jpn. J. Cancer Res (1992) 83:244-247.
  • Kimura Y, et al., “Binding of Oligoguanylate to Scavenger Receptors Is Required for Oligonucleotides to Augment NK Cell Activity and Induce IFN,” J. Biochem (1994) 116(5):991-994.
  • Kuramoto et al., “Oligonucleolide Sequences Required for Natural Killer Cell Activation,” Jpn. J. Cancer Res., (1992) 83:1128-1131.
  • Messina et al., “The Influence of DNA Structure on the in vitro Stimulation of Murine Lymphocytes by Natural and Synthetic Polynucleotide Antigens,” Cellular Immunology (1993) 147:148-157.
  • Messina et al., “Stimulation of in vitro Murine Lymphocyte Proliferation by Bacterial DNA,” The Journal of Immunology (1991) 147(6):1759-1764.
  • Mottram, et al., “a Novel CDC2-Related Protein IGnase From Leishania Mexicana.LmmCRK1. Is Post-Translationally Regulated During the Life Cycle”, J. Biol. Chem., 268(28):21044-21052 (1993).
  • Ren jun et al. (Zhonghua Zhong Zazhi, 1994, 16, 4, 247-50, HCAPLUS, AN 1995: 198874).
  • Sato et al., “Immunostimulatory DNA Sequences Necessary for Effective Intradermal Gene Immunization,” Science (1996) 273:352-354.
  • Schnell et al., “Identification and Characterization of a Saccharomyces cerevisiae Gene (PAR1) Conferring Resistance to Iron Chelators,” Eur. J. Biochem. (1991) 200:487-493.
  • Stull et al., “Antigene, Ribozyme and Aptamer Nucleic Acid Drugs: Progress and Prospects,” Pharmaceutical Research, (1995) 12(4):465-483.
  • Tanaka T. et al., An Antisense Oligonucleotide Complementary to a Sequence in IG2b Germline Transcripts, Stimulates B Cell DNA Synthesis, and Inhibits Immunoglobulin Secretion, J. Exp. Med., (1992) 175:597-607.
  • Tokunaga T. et al., “Synthetic Oligonucleotides with Particular Base Sequences from the cDNA Encoding Proteins of Mycobacterium bovis BCG Induce Interferons and Activate Natural Killer Cells,” Microbial. Immunol. (1992) 36(1):55-66.
  • Tokunaga, “A synthetic Single-stranded DNA, Poly(dG,dC), Induces Interferon-alpha/beta and—gamma, Augments Natural Killer Activity, and Suppresses Tumor Growth,” Jpn. J. Cancer Res.(1988) 79(6):682-686.
  • Wallace et al., “Oligonucleotide Probes for the Screening of Recombinant DNA Libraries” Methods in Enzymology, (1987) 152:432-442.
  • Whalen R., “DNA Vaccines for Emerging Infectious Disease: What IF?,” Emerging Infectious Disease, (1996) 2(3):168-175.
  • Wu G.Y. et al., “Receptor-mediated Gene Delivery and Expression in vivo,” J. Biological Chemistry, (1988) 263:14621-14624.
  • Yamamoto S. et al., “DNA from Bacteria, but not from Vertebrates, Induces Interferons, Activates Natural Killer Cells and Inhibits Tumor Growth,” Microbial. Immunol. (1992) 36(9):983-997.
  • Agrawal, et al., “Absorption, Tissue Distribution and In Vivo Stability in Rats of a Hybrid Antisense Oligonucleotide Following Oral Administration” Biochemical Pharmacology (1995) 50:4:571-576.
  • Agrawal, S, “Antisense Oligonucleotides: Toward Clinical Trials”, Tibtech (1996) 14:376-387.
  • Agrawal, S. and Zhang, R., “Pharmacokinetics and Bioavailability of Antisense Oligonucleotides Following Oral and Colorectal Administration in Experimental Animals” Handb. Exp. Pharmacol. (1998) vol. 131 Antisense Research and Application pp. 525-543.
  • Agrawal, S. and Zhang, R., “Pharmacokinetics of Oligonucleotides” Ciba Found Symp. (1997) 209:60-78.
  • Bodey et al. “Failure of cancer vaccines: The significant limitation of this approach to immunotherapy” pp. 2665-2676 2000.
  • Boiarkina, et al., “Dietary supplementa from ground fish meat with DNA for treatment and prophylaxis”, Vopr Pitan, (1998); (1):29-31. Abstract.
  • Brenda et al., “Immune Stimulation by an Antisense Oligomer Complementary to the rev gene of HIV-1,” Biochemical Pharmacology, (1993) 45(10):2037-2043.
  • Chace, et al., “Regulation of Differentiation in CD5+ and Conventional B Cells”, Clin. Immunol. and Immunopath, 68(3):327.332 (1993).
  • Chu, et al., “CpG Oligodeoxynucleotides Act as Adjuvants That Switch on T Helper 1 (Th1) Immunity”, J. Exp. Med., (1997) 186(10): 1623-1631.
  • Curtis, Biology, Second Edition, pp. 638-641.
  • Davis, et al., “CpG DNA Is A Potent Enhancer Of Specific Immunity In Mice Immunized With Recombinant Hepatitis B Surface Antigen”, J. Immunol, (1998) 160:870-876.
  • Doerfler, et al., “On the Insertion of Foreign DNA Into Mammalian Genomes: Mechanlsm and Consequences” Gene 157:241-245 (1995).
  • Fanslow, et al., “Effect of nucleotide restriction and supplementation on resistance to experimental murine candidiasis”, J. Parenter Enteral Nutr., (1998) 12(1):49-52 Abstract.
  • Gilboa Immunotherapy of cancer with genetically modified tumor vaccines pp. 101-107 1996.
  • Hedley et al., “Microspheres containing plasmid-encoded antigens elicit cytotocic T-cell responses” pp. 365-368, vol. 4 No. 3 1998.
  • Hohlweg et al., “On the fate of plant other foreign genes upon th uptake in food or after intramuscular injection in mice” 2001, Mol. Genet Genomics, vol. 265, pp. 225-233.
  • Jones et al. “Ploly(DdL-lactide-co-glycolide)-encapsulated plasmid DNA elicits sytemic and mucosal antibody responses to encoded protein after oral administration” pp. 814-817, vol. 15 No. 8 1997.
  • Krieg, et at, “CpG Motifs in Bacterial DNA Trigger Direct B-cell Activation”, Nature, 374:546-549 (1995).
  • Krieg, et al., “Brief Communication: Oligodeoxynucleotide Modifications Determine the Magnitude of B Cell Stimulation by CpG Motifs”, Antisense & Nucleic Acid Drug Delivery Development, 6:133-139(1996).
  • Kuchan, et al., “Nucleotides in Infant Nutrition: Effects on Immune Function” Pediatric Nutrition. Pedlatr. Adolesc. Med. Basel. Karger (1998) 8:80.94.
  • Kulkarni, et al., “Effect of dietary nucleotides on responses to bacterial infections”, J. Parenter Enteral. Nutr., (1986)10(2):169.71 Abstract.
  • Lehninger, Biochemistry, Second Edition.
  • Mastrangelo et al., “Gene Therapy for Human Cancer: An Essay for Clinicians,” Seminars in Oncology (1996) 23(1):4-21.
  • McCluskie et al. “Novel strategies using DNA for the induction of mucosal immunity” pp. 303-329 1999.
  • Perspective pp. 155-156 1999, Alton et al.
  • Ray et al. “Oral pretreatment of mice with immunostimulatory CpG DNA induces reduced susceptibility to listeria monocytogenes.” vol. 15, No. 5, pp. A1007 2001.
  • Shubbert, et al., “Ingested Foreign (phage M13) DNA Survives Transiently in the Gastrointestinal Tract and Enters the Bloodstream of Mice” Mol. Gen. Genet. (1994) 242:495-504.
  • Tortora et al. “Oral antisense that targets protein kinase a cooperates with taxol and inhibits tumor growth, angiogenesis, and growth factor production1” vol. 6, pp. 2506-2512 2000.
  • Yamamoto S. et al., “Mode of Action of Oligonucleotide Fraction Extracted from Mycobacterium bovis BCG,” Kekkaku (1994) 69(9):29-32.
  • Yamamoto S. et al., “Unique Palindromic Sequences in Synthetic Oligonucleotides Are Required to Induce IFN [correction of INF] and Augment IFN-mediated [correction of INF] Natural Killer Activity,” J. Immunol. (1992) 148(12):4072-4076.
  • Yamamoto T. et al., “Ability of Oligonucleotides with Certain Palindromes to Induce Interferon Production and Augment Natural Killer Cell Activity is Associated with their Base Length,” Antisense Res. And Devel. (1994) 4:119-123.
  • Yamamoto T. et al., “Lipofection of Synthetic Oligodeoxyribonucleotide having a Palindromic Sequence of AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity,” Microbiol. Immunol. (1994) 38(10):831-836.
  • Yamamoto T. et al., “Synthetic Oligonucleotides with Certain Palindromes Stimulate Interferon Production of Human Peripheral Blood Lymphocytes in vitro,” Jpn J. Cancer Res. (1994) 85:775-779.
  • Yew, et al., “Contribution of Plasmid DNA to Inflammation in the Lung After Administration of Cationic Lipid: pDNA Complexes” Hum Gene Ther. (1999) 20:10(2):223-234 Abstract.
  • Yew et al. “Reduced Inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs” pp. 255-262 vol. 1, No. 3 2000.
  • Press Release, Jan. 2007, “Coley Pharmaceutical Group Updates Hepatitis C Drug Development Strategy”.
  • Press Release, Jun. 2007, “Coley Pharmaceutical Group Announces Pfizer's Discontinuation of Clinical Trials for PF-3512676 Combined with Cytotoxic Chemotherapy in Advanced Non Small Cell Lung Cancer”.
  • Patent Interference No. 105,526. Krieg Substantive Motion 1 (for unpatentability based on interference estoppel). (Electronically filed, unsigned).
  • Patent Interference No. 105,526.. Krieg Substantive Motion 2 (for judgment based on inadequate written description and/or enablement). (Electronically filed, unsigned). Jun. 18, 2007.
  • Patent Interference No. 105,526. Krieg Contingent Responsive Motion (to add new claims 104 and 105). (Electronically filed, unsigned). Jul. 25, 2007.
  • Patent Interference No. 105,526. Krieg Substantive Motion 3 (for judgment based on prior art). (Electronically filed, unsigned). Jun. 18, 2007.
  • Patent Interference No. 105,526. Raz Motion 1 (Unpatentability of Krieg Claims under 35 U.S.C. § 112, First Paragraph). (Electronically filed, unsigned). Jun. 18, 2007.
  • Patent Interference No. 105,526. Raz Motion 2 (Raising a Threshold Issue of No Interference-in-Fact). (Electronically filed, unsigned). Jun. 18, 2007.
  • Patent Interference No. 105,526.. Raz Motion 3 (Krieg's Claims are Unpatentable Over Prior Art Under 35 U.S.C. § 102(b)) (Electronically filed, unsigned). Jun. 18, 2007.
  • Patent Interference No. 105,526. Raz Motion 4 (To Designate Krieg Claims 46 and 82-84 as Corresponding to Count 1). (Electronically filed, unsigned). Jun. 18, 2007.
  • Patent Interference No. 105,526. Raz Responsive Miscellaneous Motion 5 (To revive the Raz Parent Application) (Electronically filed, unsigned) Jul. 25, 2007.
  • Patent Interference No. 105,526. Raz Contingent Responsive Motion 6 (To Add a New Claim 58) (Electronically filed, unsigned) Jul. 25, 2007.
  • Patent Interference No. 105,526. Krieg Opposition 1 (Opposition to Motion for Lack of Enablement and Written Description) (Electronically filed, unsigned) Sep. 10, 2007.
  • Patent Interference No. 105,526. Krieg Opposition 2 (to Raz Motion 2) (Electronically filed, unsigned) Sep. 10, 2007.
  • Patent Interference No. 105,526. Krieg Opposition 3 (To Raz Motion 3) (Electronically filed, unsiged) Sep. 10, 2007.
  • Patent Interference No. 105,526. Krieg Opposition 4 (Opposition to Motion for Designating Claims 46 and 82-84 as Corresponding to the Court) (Electronically filed, unsigned) Sep. 10, 2007.
  • Patent Interference No. 105,526. Krieg Opposition 6 (Opposition to Raz Contingent Responsive Motion 6) (Electronically filed, unsigned) Sep. 10, 2007.
  • Patent Interference No. 105,526. Raz Opposition 1 (Opposing Krieg Substantive Motion 1) (Electronically filed, unsigned) Sep. 10, 2007.
  • Patent Interference No. 105,526. Raz Opposition 2 (Opposing Krieg Substantive Motion 2) (Electronically filed, unsigned) Sep. 10, 2007.
  • Patent Interference No. 105,526. Raz Opposition 4 (Opposing Krieg Contingent Responsive Motion to Add New Claims 104 and 105) (Electronically filed, unsigned) Sep. 10, 2007.
  • Patent Interference No. 105,526. Krieg Reply 1 (Reply to Raz opposition 1) Oct. 5, 2007.
  • Patent Interference No. 105,526. Krieg Reply 2 (Reply to Raz opposition 2) Oct. 5, 2007.
  • Patent Interference No. 105,526. Krieg Reply 4 (Reply to Raz opposition 4) Oct. 5, 2007.
  • Patent Interference No. 105,526. Raz Reply 1 (Reply to Krieg opposition 1) Oct. 5, 2007.
  • Patent Interference No. 105,526. Raz Reply 2 (Reply to Krieg opposition 2) Oct. 5, 2007.
  • Patent Interference No. 105,526. Raz Reply 3 (Reply to Krieg opposition 3) Oct. 5, 2007.
  • Patent Interference No. 105,526. Raz Reply 4 (Reply to Krieg opposition 4) Oct. 5, 2007.
  • Patent Interference No. 105,526. Raz Reply 6 (Reply to Krieg opposition 6) Oct. 5, 2007.
  • Patent Interference No. 105,526. Krieg Miscellaneous Motion 5 (To exclude exhibits 2066, 2070, 2071, 2072, 2073, 2074, 2075, 2076 and 2078) Oct. 9, 2007.
  • Patent Interference No. 105,526. Raz Opposition 5 (Opposing Krieg Miscellaneous Motion 5) Oct. 25, 2007.
  • Patent Interference No. 105,526. Raz Miscellaneous Motion 7 (To exclude evidence) Oct. 19, 2007.
  • Patent Interference No. 105,526. Krieg Opposition 7 (To Raz Miscellaneous Motion 7) Oct. 25, 2007.
  • Patent Interference No. 105,526. Krieg Reply 5 (Reply to Raz opposition 5) Oct. 30, 2007.
  • Patent Interference No. 105,526. Raz Reply 7 (Reply to Krieg opposition 7) Oct. 30, 2007.
  • Patent Interference No. 105,526. Order—Bd.R. 104. Conference Call. Paper 211. Sep. 30, 2008.
  • Patent Interference No. 105,526. Memorandum Opinion and Order (Decision on Motions) Dec. 1, 2008.
  • Patent Interference No. 105,526. Judgment on Preliminary Motions under 37 C.F.R §41.127 Dec. 1, 2008.
  • Patent Interference No. 105,526. Paper 217. Raz Notice of Filing of a Notice of Appeal (Appeal to the Court of Appeals for the Federal Circuit). Jan. 27, 2009.
  • Patent Interference No. 105,526. Paper 218. Raz Notice of Withdrawal of Appeal. May 15, 2009.
  • Patent Interference No. 105,674. Paper No. 1. Declaration under 37 C.F.R. §41.203(b) Dec. 1, 2008.
  • Patent Interference No. 105,674. Paper No. 6 Raz Notice of Real Party in Interest. Dec. 12, 2008.
  • Patent Interference No. 105,674. Paper No. 11 Krieg Designation of Real Party in Interest. Dec. 15, 2008.
  • Patent Interference No. 105,674. Paper No. 15. Order—Bd.R. 104(c) Summary of Dec. 23, 2008 Conference Call.
  • Patent Interference No. 105,674. Paper No. 19. Order—Bd.R. 104(c). Conference Call. Jan. 16, 2009.
  • Patent Interference No. 105,674. Paper No. 21. Raz Observations (regarding evidence to support certain proposed motions. Jan. 27, 2009.
  • Patent Interference No. 105,674. Paper No. 23. Raz Miscellaneous Motion 1 (to revive the Raz parent application). Jan. 27, 2009.
  • Patent Interference No. 105,674. Paper No. 25. Order—Bd.R. 104(c) (Raz v. Krieg) Summary of Conference Call on Feb. 4, 2009.
  • Patent Interference No. 105,674. Paper No. 29. Joint Submission Pursuant to Order Dated Jan. 16, 2009. Mar. 11, 2009.
  • Patent Interference No. 105,674. Paper No. 32. Raz Abandonment of Contest. May 15, 2009.
  • Patent Interference No. 105,674. Paper No. 33. Judgment—Bd.R. 127. May 20, 2009.
Patent History
Patent number: 8309527
Type: Grant
Filed: Feb 26, 2004
Date of Patent: Nov 13, 2012
Patent Publication Number: 20040152657
Assignees: University of Iowa Research Foundation (Iowa City, IA), The United States of America, as represented by the Secretary, Department of Health and Human Services (Washington, DC), Coley Pharmaceutical Group, Inc. (New York, NY)
Inventors: Arthur M. Krieg (Wellesley, MA), Dennis Klinman (Potomac, MD), Alfred D. Steinberg (Potomac, MD)
Primary Examiner: N. M Minnifield
Attorney: Wolf, Greenfield & Sacks, P.C.
Application Number: 10/789,536